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O ce
Dipartimento di Scienze Biomediche Sperimentali e Cliniche
Dottorato di Ricerca in Biochimica e Biologia Applicata
XXV Ciclo
Coordinatore del Dottorato: Prof.ssa Donatella Degl’Innocenti
NUTRITIONAL AND HORMONAL MODULATION
OF HUMAN MELANOMA PROGRESSION
Candidato Tutor
Dr.ssa Valentina Farini Prof. ssa Paola Chiarugi
SETTORE SCIENTIFICO DISCIPLINARE: BIO/10
ANNI 2010/2012
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3
INDEX
ABBREVIATIONS ..................................................................................................................... 6
INTRODUCTION ..................................................................................................................... 10
THE TUMOUR MICROENVIRONMENT ....................................................................... 10
Fibroblasts ....................................................................................................................... 10
Tumour microenvironment and cancer progression ....................................................... 17
MELANOMA ........................................................................................................................ 23
Definition ........................................................................................................................ 23
Etiology ........................................................................................................................... 23
Incidence ......................................................................................................................... 24
Mortality ......................................................................................................................... 24
Prevalence ....................................................................................................................... 25
Risk Factors .................................................................................................................... 25
Clinical Aspects .............................................................................................................. 26
Histopathologic factors and prognosis ............................................................................ 29
Medical treatment ........................................................................................................... 29
Biological and genetic factors ......................................................................................... 31
STRESS HORMONES: EPINEPHRINE AND NOREPINEPHRINE ............................ 33
Definition of stress .......................................................................................................... 33
The adrenal gland ............................................................................................................ 34
The periferic nervous system and the hypothalamus-pituitary-adrenal axis (HPA) ...... 36
Biosinthesis of catecholamines ...................................................................................... 41
Release and action of catecholamines ............................................................................. 43
The adrenergic receptors ................................................................................................. 47
Catecholamines and cancer ............................................................................................. 51
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HYPOXIA AND HYPOXIA INDUCIBLE FACTORS (HIFs) ........................................ 57
HIF-1α (Hypoxia-Inducible Factor-1α) .......................................................................... 60
HIF-2α (Hypoxia-Inducible Factor-2α) .......................................................................... 61
HIF-3α (Hypoxia-Inducible Factor-3α) .......................................................................... 64
Regulation of HIFs expression ........................................................................................ 65
HIFs target genes ............................................................................................................ 79
THE METABOLISM OF CANCER CELLS ..................................................................... 89
Glycolisis ........................................................................................................................ 89
The “Warburg Effect” ..................................................................................................... 96
Molecular regulators of cancer cells metabolism ............................................................ 99
Models of metabolic symbiosis..................................................................................... 106
MATERIALS AND METHODS ........................................................................................... 111
MATERIALS ...................................................................................................................... 111
Common use solutions .................................................................................................. 112
METHODS .......................................................................................................................... 113
Cell cultures and treatments .......................................................................................... 113
Isolation of human dermal fibroblasts........................................................................... 113
Preparation of conditioned media ................................................................................. 114
Cell lysis and protein quantification ............................................................................ 114
SDS-PAGE analysis ...................................................................................................... 115
Western Blotting ........................................................................................................... 116
Crystal violet proliferation assay .................................................................................. 117
Annexin V/Iodidium Propide cytofluorimetric staining ............................................... 118
Intracellular ROS evaluation ......................................................................................... 118
Cell transfection with lipofectamine ............................................................................. 119
Invasion assay ............................................................................................................... 119
Real-time PCR .............................................................................................................. 120
Quantitative MMPs activity assay ................................................................................ 120
Statistical analysis ......................................................................................................... 121
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EXPERIMENTAL PART I ................................................................................................... 122
AIM OF THE STUDY I ......................................................................................................... 123
RESULTS I .............................................................................................................................. 125
Preliminary results: expression of β-ARs in tissue samples ......................................... 125
β-AR expression analysis and responsiveness of melanoma cell lines after
catecholamines stimulation ........................................................................................... 128
Catecholamines increase melanoma cells invasive ability. ........................................... 133
Effects of catecholamines on cytokine production and tumour microenvironment. ..... 138
DISCUSSION I........................................................................................................................ 143
CONCLUDING REMARKS I ............................................................................................... 147
EXPERIMENTAL PART II .................................................................................................. 149
AIM OF THE STUDY II ........................................................................................................ 150
RESULTS II ............................................................................................................................ 152
Role of nutrients in regulating melanoma cells proliferation and survival ................... 152
Nutrients promote normoxic HIF-1α and CA-IX expression and affect ROS production.
...................................................................................................................................... 158
Nutrients promote CA-IX expression in a HIF-1α-dependent manner ......................... 162
Nutrients-induced HIF-1α expression promotes melanoma cells invasiveness ............ 165
DISCUSSION II ...................................................................................................................... 169
CONCLUDING REMARKS II ............................................................................................. 172
BIBLIOGRAPHY ................................................................................................................... 174
APPENDIX: PUBLICATIONS ............................................................................................. 212
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ABBREVIATIONS
5-HETE: 5-Hydroxyeicosatetraenoic acid
5-LOX: 5-llipoxygenase
AA: arachidonic acid
AAAD: aromatic L-amino acid decarboxylase
ACTH: adrenocorticotropic hormone
Akt: serin-threonin protein kinase
AMF: autocrine motility factor
AP1: activating protein 1
APAF1: apoptotic protease activating factor 1
AR: adrenergic receptors
ARE: antioxidant responsive element
ATP: adenosine triphosphate
AVP: arginin-vasopressin protein
BAD: Bcl-2-associated death promoter
Bcl2: B cells lymphoma 2
CA: catecholamines
cAMP: cyclic AMP
COX-2: cyclooxygenase type 2
CRH: corticotropic hormone
CSF: colony stimulating factor
DA: dopamine
DAG: diacylglycerol
DBH: dopamine hydroxylase
DOPA: L-3,4-dihydroxyphenylalanin
E: epinephrine
ECM: extracellular matrix
EGF: endotelial growth factor
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EMT: epithelial-mesenchimal transition
EPAC: exchange protein directly activated by cAMP
EPO: erythropoietin
ERK: extracellular-signal-regulated kinases
FADH: flavin adenine dinucleotide
FAK: focal adhesion protein
FGF: fibroblasts growth factor
FGFR: fibroblasts growth factor receptor
FH: fumarase
FIH: factor inhibiting HIF-1
FSP1: fibroblast specific protein-1
Gab 1: GRB2-associated-binding protein 1
G-CSF: granulocyte colony stimulating factor
GLUT1: glucose transporter 1
GLUT3: glucose transporter 3
GM-CSF: granulocyte-macrophage colony stimulating factor
Grb2: growth factor receptor-bound protein 2
GREs: glucocorticoids responsive elements
GSH: gluthation
GTP: guanosine triphosphate
HDF: human dermal fibroblasts
HGF: hepatocyte growth factor
HIF: hypoxia inducible factor
HPA axis: hypotalamic-pituitary-adrenal axis
HREs: hypoxia responsive elements
Hsp 90: heat shock protein 90
IFN-γ: interferon-γ
IFP: pression of interstitial fluid
IGF: insulin-like growth factor
IL-1b, 4, 6, 8, 10, 12, 13, 23: interleukin 1b, 4, 6, 8, 10, 12, 13, 23.
8
IPAS: inhibitory PAS domain protein
JNK: N-terminal jun kinase
LDH-A: lactate dehydrogenase A
LPS: lipopolisaccharyde
LT: leucotriens
MAPK: mitogens-activated protein kinase
MCT-1, MCT-4: monocarboxylate transporter type 1 and 4
MF: myofibroblasts
MMPs: matrix metalloproteinase
NAC: N- acetyl cystein
NADH: nicotinamide adenin dinucleotide
NADPH: nicotinamide adenin dinucleotide phosphate
NE: norepinephrine
NF-kB: nuclear factor kB
NO: nitric oxide
Oct4: octamer-binding transcription factor 4
ODDD: oxygen-dependent degradation domain
PAF: platelet-activating factor
PDGF: platelet-derived growth factor
PDGFR: platelet-derived growth factor receptor
PDK: pyruvate dehydrogenase kinase
PGD2: prostaglandin D2
PGH2: prostaglandin H2
PHD: prolil-hydroxylase
PI3K: phosphoinositide 3-kinase
PKA: protein kinase A
PKB/AKT: protein kinase B or AKT
PKC: protein kinase C
PKM2: pyruvate kinase isoform M2
PLC: phospholipase C
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PNMT: phenylethanolamine n-methyltransferase
PTP: protein tyrosine phosphatase
pVHL: von Hippel Lindau protein
ROI: reactive oxygen intermediates
ROS: reactive oxygen species
RTK: tyrosine-kinase receptor
SDF-1: stromal derived factor 1
SDH: succinate dehydrogenase
SIRT: sirtuin
SOD: superoxyde dismutase
STAT: signal transducer and activator of transcription
TGF-β: transforming growth factor-β
TH: tyrosine hydroxylase
TNF-α: tumor necrosis factor α
TPA: 12-O-tetradecanoylphorbol-13-acetate
TRX: thyoredoxin
U1, U2: uptake type 1 and 2
uPA: urinary plasminogen activator
uPAR: urinary plasminogen activator receptor
VEGF: vascular endothelial growth factor
VEGFR: vascular endothelial growth factor receptor
α-SMA: α-smooth muscle actin
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INTRODUCTION
THE TUMOUR MICROENVIRONMENT
Tissues contain several cell types that work synergically in order to regulate normal
physiology. These cells have positional identity so that their location and number are
strictly defined. Cancer cells have lost these constrictions, but they keep creating a
complex and continuative “cross-talk” with surrounding, non-malignant cells and/or
with the extracellular architecture made of direct cell-to-cell contacts and
paracrine/exocrine signals. These interactions are not static, but they evolve along with
tumour progression. The tumour microenvironment can exert an inibhitory effect on
malignant cells aggressiveness, but a state of constant alteration of microenvironment
itself can become in turn strongly tumour-growth promoting. In fact, during the early
stages of cancer development, the protective constraints of the microenvironment are
overridden by conditions such as chronic inflammation and the local tissue
microenvironment shifts to a growth-promoting state. In this light fibroblasts represent
the key mediators in promoting tumour progression (Joyce e Pollard, 2009).
Fibroblasts
Fibroblasts are elongated cells, characterized by extensive cellular processes, with
fusiform and tapered shape (Tarin and Croft 1969). They can be easily isolated from
tissues and cultured in vitro. Their fusiform morphology makes them identifiable and,
despite the paucity of specific markers, some molecules can be related to a fibroblastic
phenotype, although none of these is exclusive of fibroblasts and/or expressed in all
fibroblasts. Among these markers, the fibroblasts specific protein-1 (FSP-1) seems to
provide the best specificity for the identifications of fibroblasts in vivo, but other
markers can be considered site-specific, like desmin, a specific marker for skin
fibroblasts.
A recent study has demonstrated high fibroblasts heterogeneity in mammals as they
show completely different gene expression pattern in anatomically different tissues.
This difference is evident from the specific secretion of extracellular matrix (ECM)
factors, growth or differentiation factors. For example, fetal skin fibroblasts express
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genes encoding for collagen type I and V, while those from lung tissues express
different collagen patterns (Chang H. Y. et al. 2002).
Fibroblasts functions include ECM deposition, regulation of epithelial differentiation,
wound healing and inflammatory mechanisms (Tomasek et al. 2002; Parsonage et al.
2005). According to that, fibroblasts produce several fibrillar ECM components like
fibronectin and collagen type I, III and V (Tarin and Croft 1969; Tomasek et al. 2002).
In particular, fibroblasts actively contribute to basal membrane formation through
laminin and collagen type IV deposition. In addition, fibroblasts secrete proteases of the
metalloproteinase (MMPs) family, which have a crucial role in regulating ECM
turnover, and tissues homeostasis through growth factors production and regulation of
epithelial cell-to-cell cross-talk (Simian et al. 2001; Chang et al. 2002).
Healthy fibroblasts are located within the ECM of connective tissue and constitutively
express vimentin and FSP-1. As a result of specific environmental stimuli, fibroblasts
may undergo an activated state, named “myofibroblastic”, which is characterized by the
de novo expression of α-SMA protein, the actin isoform typical of smooth muscle cells,
and the ability to synthetize large amounts of collagen and components of the ECM.
Unlike normal fibroblasts which contains a well-developed rough endoplasmic
reticulum, myofibroblasts are characterized by a large eucromatinic nucleus with two
nucleoli, a prominent Golgi apparatus, and express microfilaments with dense bodies
similar to those found in smooth muscle cells. In particular, myofibroblasts play a very
important role in regulating tumour progression through three main mechanisms:
Expression of ECM specific components;
Activation of remodelling mechanisms in the granulose tissue and tension
transmission to cancer cells through a Rho/Rho-kinase-dependent pathway;
Cytokines secretion.
During tissutal lesion, different stimuli induce the activation of fibroblasts like growth
factors such as the transforming-growth factor β (TGF-β), epidermal growth factor
(EGF), platelet-derived growth factor (PDGF) and b-fibroblasts-growth factor (bFGF),
produced by both the same damaged epithelial cells and the infiltrated mononuclear
cells, such as monocytes and macrophages (Kalluri and Zeisberg, 2006; Orimo and
Weinberg, 2006). This activation can also occur through direct cell-cell interaction by
binding with leukocytes through adhesion molecules like intercellular adhesion
molecule-1 (ICAM1) and the vascular cell adhesion molecule-1 (VCAM1), or through
reactive oxygen species (ROS) and complement C1 factor (Clayton et al. 1998) (Fig. 1).
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Fig. 1 Main characteristics of activated and not-activated fibroblasts.
Myofibroblasts are able to secrete higher levels of proteases that degrade the ECM, such
as MMP-2, MMP-3, MMP-9, thereby increasing the turnover and altering the
composition of the ECM. In addition, the activated form of fibroblasts produces growth
factors like hepatocyte growth factor (HGF), insulin-like growth factor (IGF), nerve
growth factor (NGF), WNT1, epidermal growth factor (EGF), and FGF-2, also
promoting adjacent epithelial cells proliferation (Bhowmick et al. 2004).
Beside their expression in sclerotic tissue and sites of wound repair, activated
fibroblasts act as modulators of the immune response through the secretion of cytokines,
such as interleukin-1, and chemokines. In the case of wound repair, myofibroblasts
return in the idle state once the stimulus is attenuated, while in fibrosis and cancer
fibroblasts keep an activated state until the death of the surrounding tissue. In fact,
fibroblasts isolated from a fibrotic tissue maintain their activated phenotype in vitro, as
demonstrated by their continuous expression of ECM constituents, growth factors and
cytokines. These observations show a self-maintaining autocrine and paracrine
signaling that stimulates other fibroblasts. However, mechanisms underlying this
continuous activation are not yet clearly understood (Strieter et al. 1989; Rollins et al.
1989).
In the early stages tumour is considered as a carcinoma in situ where tumour cells form
a neoplastic lesion which spreads in the microenvironment of a target tissue, but
remaining separate from the surrounding tissue as it turns out to be contained within the
borders of basement membrane. Carcinoma in situ is associated with a stroma similar to
13
that observed during wound repair, and is therefore defined as "activated or reactive
stroma” (Hanahan and Weinberg, 2000; Dvorak et al. 1984; Ronnov-Jessen et al.,
1996).
There are some key differences between normal and activated stroma: the first one
contains a minimum number of fibroblasts associated with a physiological ECM
(Ronnov-Jessen et al., 1996), while the second is associated with an increase of
fibroblasts, capillaries and deposition of type I collagen and fibrin. In particular,
activated vascular endothelial growth factor (VEGF), produced mainly by fibroblasts
and inflammatory cells, represents a key molecule for the development of the stroma
(Brown et al. , 1999). VEGF induces microvascular permeability, thus allowing the
extravasation of plasma proteins such as fibrin, which attracts fibroblasts, endothelial
cells and inflammatory cells (Senger et al. 1983; Dvorak et al. 1991; Brown et al. 1999).
These cells produce ECM that is rich in fibronectin and type I collagen, both implicated
in the development of tumour angiogenesis (Leung et al. 1989; Brown et al . 1999; Feng
et al. 2000).
During tumour progression from carcinoma in situ to invasive carcinoma tumour cells
invade the reactive stroma (Dvorak 1986; Ronnov-Jessen et al. 1996). Basement
membrane and stroma are degraded, and myofibroblast come into direct contact with
the tumour cells. Invasive cancer is usually associated with the expansion of tumour
stroma and to an increase of the deposition of ECM, known as desmoplasia (Shekhar et
al. 2003; Ronnov-Jessen et al. 1996; van Kempen et al. 2003; Schedin and Elias 2004).
This phenomenon appears to be very similar to the changes that take place during
fibrosis, but while fibrosis is associated with a decrease of vascularization (Brown et al.
1989), solid tumours are more vascularized than healthy tissues (Folkman et al. 1989).
The desmoplastic stroma contains a larger amount of fibrillar collagen, fibronectin,
proteoglycans and tenascin C (Ronnov-Jessen et al. 1996). In particular, tenascin C is
absent in mature mammary glands, but is highly expressed in breast tumours (Chiquet -
Ehrismann et al. 1986; Mackie et al. 1987) and correlated with increased invasiveness
and a worse prognosis (Ishihara et al. 1995; Brunner et al. 2004; Mackie et al. 1987; De
Wever and Mareel 2002).
The activated fibroblasts within the tumour stroma are called Cancer Associated
fibroblasts (CAFs) (Barsky et al. 1984; De Wever and Mareel, 2002; Mueller and
Fusenig, 2004) and appear as large mesenchymal cells with tapered shape and with
similar characteristics to smooth muscle cells and fibroblasts. They were first identified
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by immunocytochemistry using a combination of different markers such as α-SMA (α-
smooth-muscle-actin), vimentin, desmin and fibroblast activation protein (FAP), a
serine protease located on the cell surface of tumour stromal fiboblasts (Garin-Chesa et
al. 1990; Lazard et al. 1993; Mueller and Fusenig 2004).
The presence of CAFs in the activated tumour stroma has been observed in various
types of cancer like breast cancer (Chauhan et al. 2003), prostate cancer (Olumi et al.
1999; Mueller and Fusenig 2004) and skin tumours (Skobe and Fusenig 1998). In fact,
it has been demonstrated in vivo that the combination of prostatic epithelial cells with
healthy CAFs leads to a limited tumour growth that resembles a prostatic intraepithelial
neoplasia, while injecting them with prostatic epithelial non-tumourigenic immortalized
cells results in the formation of malignant tumours (Olumi et al., 1999; Cunha et al.,
2003). CAFs induce tumourigenic alterations in epithelial cells and promote tumour
progression through specific interactions with tumour cells, promoting their
invasiveness if co-injected into mice models (Dimanche-Boitrel et al. 1994). CAFs are
also able to express various growth factors and cytokines like IGF-1 and HGF that
promote survival, migration and invasion of tumour cells (Li et al. 2003; De Wever et
al. 2004; Lewis et al. 2004). Through expression of growth factors like VEGF or MCP-
1, CAFs contribute to tumour microenvironment formation by activating tumour stroma
through stimulation of angiogenesis and recruitment of inflammatory cells (Orimo A. et
al., 2005; Erez N. et al., 2010). In addition, they also produce proteases such as MMPs
able to degrade the ECM allowing the cancer cells to overcome the boundaries of the
tissues and therefore to exit from the primary site of the tumour (Sternlicht et al. 1999;
Boire et al. 2005). MMPs and other proteases act directly on the motility and
invasiveness of cancer cells. This was demonstrated for MMP-3, also known as
stromelysin-1, a MMP highly expressed in fibroblasts that cuts the extracellular domain
of E-cadherin, thus promoting epithelial-mesenchymal transition (EMT) and increasing
tumour cells invasiveness (Lochter et al. 1997) (Fig. 2).
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Fig. 2. Cross-talk between stromal fibroblasts and different types of cells of tumour
microenvironment.
Tumour cells release growth factors such as TGF-β, PDGF and bFGF that mediate the
activation of fibroblasts (Dvorak 1986; Elenbaas and Weinberg 2001). TGF-β is
associated with an increase of fibrotic tissue, tumour progression and fibroblasts
recruitment (Siegel and Massague 2003). In addition, TGF-β is the most important
factor of the tumour microenvironment promoting EMT in tumour cells and leading
fibroblasts to a CAF phenotype in vitro (Siegel and Massague 2003). In healthy tissue,
TGF-β impairs growth of epithelium and normal development of malignant tumours
(Hanahan and Weinberg 2000; Siegel and Massague 2003), therefore the TGF-β
probably has a role as tumour suppressor (Akhurst 2002). For what fibroblasts is
concerned, TGF-β facilitates interactions with epithelial cells that suppress the onset of
cancer (Bhowmick et al. 2004). On the contrary, in advanced stages of tumour
progression TGF-β progressively loses its anti-proliferative effects while facilitates
EMT programme in tumour cells, thereby increasing invasiveness and metastatization
(Siegel and Massague 2003). The signaling pathways that are involved in TGF–β-
mediated EMT involve Smad proteins, in particular SMAD3, the phosphatidylinositol-
16
3-kinase-Akt and p38 MAPK pathways (Derynck et al., 2001; Kalluri and Neilson
2003). Also PDGF is correlated with tumour progression (Bronzert et al., 1987),
although most of tumour cells do not express its receptor (Forsberg et al. 1993). Unlike
TGF-β, PDGF induces fibroblasts proliferation, but not the acquisition of the activated
phenotype associated to an excessive deposition of ECM (Shao et al. 2000). bFGF is
another growth factor that stimulates the proliferation of fibroblasts, but not the
expression of α-SMA (Armelin 1973; Ronnov - Jessen et al. 1996).
Since EMT is a phenomenon whereby epithelial cells lose their cell-cell contacts
acquiring mesenchymal properties, it promotes the onset of a more invasive phenotype
(Hay 1995). The EMT of healthy epithelial cells adjacent to tumour cells contributes to
the development of CAFs (Kalluri and Neilson 2003). Recent studies on lung epithelial
cells have provided evidence of transition of these cells to fibroblasts during pulmonary
fibrosis development, suggesting that EMT is an important source of fibroblasts (Robin
1978). Furthermore it has been shown that exposure of cells to epithelial MMPs may
lead to increased ROS cellular levels, which stimulate the transdifferentiation into
myofibroblasts. These studies suggest that the increased expression of MMPs can
stimulate fibrosis, tumourigenesis and tumour progression through induction of a
specific EMT programme in which epithelial cells directly differentiate into activated
myofibroblasts (Radisky et al . 2007).
It has been observed that the level of TGF-β on fibroblasts, through a PKC-dependent
signaling, induces a significant increase in ROS levels that appear to be key secondary
messengers of the signaling pathway triggered by TGF-β (Cat B. et al. 2006).
According to that, treatment with antioxidants such as NAC or selenite causes an almost
complete regression of the TGF-β signaling pathway, thereby blocking the expression
of α-SMA and the transdifferentiation to myofibroblasts. One of the major effects
induced by TGF-β stimulation is a significant increase in lipid hydroperoxides.
Blocking this stimulation there is a strong inhibition of the TGF-β-induced fibroblasts
activation, thus suggesting that lipid hydroperoxides are the main messengers
responsible for the redox-dependent signal transduction triggered by TGF-β . It was also
demonstrated that following treatment with TGF-β and subsequent fibroblasts activation
there is significant gene expression changes, including induction of α-SMA and
cytokines such as HGF, VEGF and interleukin 6 (IL-6). These changes are typical of
myofibroblasts as they influence tumour cells invasive capacity (Cat B. et al. 2006).
17
It should be noted that, although it is widely accepted that CAFs assume a
myofibroblastic phenotype, the in vivo mediators are not well understood yet (Kalluri
and Zeinberg, 2006). Only some cytokines like TGF-β released by tumour cells have
been associated with the differentiation of fibroblasts (Kalluri and Zeisberg 2006;
Massague J. 2008). Also recent studies conducted in our laboratory have shown that IL-
6, secreted by prostate carcinoma PC3 cells isolated from a bone metastasis of prostate
carcinoma cells (PCa), promotes a particular phenotype named PCa-activated fibroblast
(PCa-AF). In contrast to the TGF-β-dependent phenotype, these cells do not express α-
SMA, but their activated state is confirmed by the expression of the FAP protein and
production of ECM. The PCa-AFs strongly activate the process of EMT and therefore
PC3 cells invasiveness as demonstrated by the fact that interruption of IL-6-mediated
signaling between PC3 and fibroblasts impairs the process (Giannoni et al., 2010).
According to our observations, production of IL-6 by tumour cells has been correlated
to higher carcinomas aggressiveness (Royuela M et al., 2004; Chung LW et al., 2005;
YN Niu et al., 2009).
It has been also demonstrated that CAFs are composed by a mixed population of both
-SMA positive fibroblasts (MF), probably activated by a TGF--dependent
mechanism, and PCa-AF, -SMA negative fibroblasts activated by an IL-6-dependent-
mechanism, which suggests a coexistence in vivo of phenotypes reliable to both
myofibroblasts and PCa-AFs. These data lead to the hypothesis that different
populations of CAFs have specific roles in tumour growth. Both types of fibroblasts are
able to induce EMT in PC3 cells and a greater ability invasive, involving different
pathways like urinary plasminogen activator (uPA/uPAR) for the phenotype MF and the
MMPs for the phenotype PCa-AFs (Giannoni et al., 2010). Thus endothelial cells lose
cell-to-cell contact and acquire mesenchymal properties, thereby showing greater
invasive and metastatic abilities (Hay 1995; Thiery 2006).
Tumour microenvironment and cancer progression
A raising number of evidences have demonstrated a strong, positive correlation between
cancer and tissue microenvironment. Tumour progression is considered as the result of
constant communication between cancer cells and the surrounding stroma, also called
“tumour reactive stroma”, composed by a specific ECM and several heterotypic cells
like, proinflammatory cells (cancer-associated macrophages, CAMs), endothelial cells
and perycites. This interplay has a fundamental role in different aspects of tumour
18
biology. Tumour stroma composition can vary among different kind of tumours and it is
not directly correleated to its aggressiveness. Anyway, recent studies in vivo have
shown that tumour-derived stromal fibroblasts are crucial for breast cancer development
(Dvorak et al., 1983; Kuperwasser et al., 2004).
As previously stated, this “tumour reactive stroma” can also be morphologically defined
as “a desmoplastic response”, referring to growth of dense connective tissue rich in
fibroblasts, proinflammatory and immune cells which produce large amounts of
collagen, fibronectin, proteoglycans and tenascin C (Ronnov-Jessen et al., 1996) (Fig.
3). A desmoplastic microenvironment can support key cancer mechanisms like
development of new blood and lymphatic vessels in order to favor cancer growth and
dissemination (Folkman 2003).
Fig. 3 Cancer development: a multi-step process which requires activation of the surrounding
stroma.
Cancer cells create an hospitable microenvironment producing stroma-modulating
factors like bFGF, PDGF, EGF, colony-stimulating factor (CSF) and TGF-β. These
factors activate surrounding cells like fibroblasts, smooth muscle cells and adypocites,
both in a paracrine and autocrine way, which in turn produce new cytokines, growth
factors and proteolytic enzymes responsable of ECM remodelling and cancer cells
19
invasion (Coussens and Werb, 2002; Bergers and Benjamin, 2003; Stetler-Stevenson
and Yu 2001; Mueller et al. 2003).
The mechanism of ECM degradation also induces unmasking of proteins cryptic domains
(Kalluri 2003). For example, matrix MMPs can partially degrade molecules on cells
surface and soluble factors during ECM remodelling in order to create new activated
factors with pro-migratory and proangiogenic abilities (Brinckerhoff and Matrisian 2002;
McCawley and Matrisian 2001; Egeblad and Werb 2002). In particular, cancer
aggressiveness is often positively correlated to expression levels of MMP-1 and MMP-
9. Cancer cells co-coltured with stromal cells and injected in murine models express
larger amounts of MMP-1 and MMP-9 in comparison with benign cancer cells
(Borchers et al. 1997; Airola e Fusenig 2001; Egeblad e Werb 2002).
The proteolytic fragments produced by cancer cells proteases can also modulate
proliferation, survival and migration of surrounding endothelial cells. In particular, the
action of several types of MMPs like MMP-3, MMP-7, MMP-9, MMP-12 can generate
angiostatin from the amino-terminal part of plasminogen. Other anti-angiogenic factors
are endostatin, proteolytic fragment from collagen type VIII reducing VEGF-mediated
growth and motility of endothelial cells, and tumstatin, generated from collagen IV
digestion, which is an endothelial proapoptotic factor (Dong et al. 1997; Cornelius et al.
1998O’Reilly et al.1997; Marneros e Olsen 2001). In conclusion, the regulation of
proteases homeostasis by cancer cells represents a crucial step for tumour angiogenic
ability, which results from a continuative balance between pro and antiangiogenic
signals.
Cancer cells also produce proinflammatory cytokines in order to recruite hematopoietic
cells like lymphocytes, monocytes/macrophages and neutrofils nearby the tumour mass
(Bachmeier et al. 2000; Coussens e Werb 2002). CSF-1 induces growth and
differentiation of mononucleated cells like macrophages and is highly expressed in
breast, ovaric and prostate cancers (Kacinski 1995). Loss of CSF-1 expression prevents
macrophages recruitment by epithelial cancer cells and reduces tumour growth and
metastatization. (Lin et al. 2001).
Recent studies have demonstrated the importance of hematopoietic factors like
granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony
stimulating factor (GM-CSF) which promote mobilization of monocyte/macrophages
and neutrofils from bloodstream and surrounding tissues. Growing proinflammatory
signals can in turn induce recruitment of endothelial cells progenitors, thereby
20
promoting angiogenesis into the tumour burden. Neutrofils ease this process
remodelling the ECM through secretion of MMP-9 and MMP-13. Also macrophages
express several types of proteases like the uPA and MMPs which generate pro-
angiogenic molecules from ECM degradation like VEGF (Obermueller et al. 2004;
Carmeliet e Jain 2000) (Fig. 4).
Fig. 4 The interplay between tumour reactive stroma and endothelial cells.
In vivo studies have shown that blocking antibodies against VEGF receptor 2
(VEGFR2) impairs angiogenesis and cancer invasive spur, inducing a regression from a
high malignant cancer to a pre-malignant, not invasive phenotype. This process is
strongly correlated to stabilization of the tumour stroma which turns into a normal
connective tissue with new epithelial structures, like intact membranes basements and
cell-to-cell junctions, and well-organised collagen fibres nearby the tumour.
Normalization of tumour stroma is probably correlated to impairment of MMPs activity.
As a result the reduced turnover of laminin, fiboronectin and collagen type I and IV
promotes their accumulation and correct reorganization, thereby reducing angiogenensis
21
and almost completely reverting cancer progression. (Skobe et al 1998; Vajkoczy et al.
2000) (Fig. 5).
Fig. 5 Tumour-activated stroma normalization induces tumour regression.
In the light of these experimental evidences, tumour microenvironment could be
considered a promising therapeutic target because tumour stroma cells are genetically
more stable than cancer cells and show less ability in developing drug resistance
(Kerbel 1997; Sporn e Suh 2000). For example, the inhibition of cytokine production by
inflammatory cells after administration of non-steroidal drugs impairs the risk of
developing colon and breast cancer (Ricchi et al. 2003). In addition, MMP inhibitors are
proving to be promising antitumour agents in the clinic. Inactivation of MMPs by
administration of synthetic MMP inhibitors may prevent initiation of MMP activation
cascades and excessive ECM degradation. On the other side, these small molecule
inhibitors also exhibit unpleasant side effects due to their broad substrate specificity.
The lack of highly specific MMP inhibitors for individual MMPs makes it difficult to
understand the role of individual MMPs. In addition, cancer cells can also avoid
22
farmacological treatment adopting an ameboid, MMPs-independent motogenic program
(Stetler-Stevenson e Yu 2001).
The tumour stroma can also act on cancer cells as a mutagenic factor itself. Genomic
instability is a commonly observed feature of tumours. Most investigations addressing
the mechanism of tumour progression have focused on the genetic factors that may play
a role. Growing evidence now suggests that, in addition to these endogenous factors, the
exogenous environment within solid tumours may by itself be mutagenic and constitute
a significant source of genetic instability. The tumour microenvironment is
characterized by regions of fluctuating hypoxia, low pH, and nutrient deprivation. Each
of these microenvironmental factors has been shown to cause severe disturbance in cell
metabolism and physiology. Both in vivo and in vitro data demonstrate that exposure of
tumour cells to adverse conditions can directly cause mutations, contributing to genetic
instability (Yuan and Glazer 1998).
23
MELANOMA
Definition
Melanoma is the typical malignant tumour of melanocytes, cells that secrete a dark
pigment, melanin, which is responsible for the color of skin. They are predominantly
located in the epidermal basal layer, but are also found in other parts of the body, like
the bowel, the oral cavity, the inner ear, the meninges and the eyes, so melanoma can
originate in any part of the body that contains melanocytes. Cutaneous melanoma
represents only the 3-5% of skin cancers, but it causes the majority (75%) of deaths
related to skin cancer because of its high invasive and metastatic ability (Boring et al.,
1991).
Etiology
The earliest stage of melanoma starts when the melanocytes begin to grow out of
control. Melanocytes are found between the outer layer of the skin (the epidermis) and
the next layer (the dermis). This early stage of the disease is called the radial growth
phase, and the tumour is less than 1mm thick. Because the cancer cells have not yet
reached the skin blood vessels, it is very unlikely that this early-stage cancer will spread
to other parts of the body. If the melanoma is detected at this stage, then it can usually
be completely removed with surgery. When the tumour cells start to move vertically up
into the epidermis and into the papillary dermis, the behaviour of the cells changes
dramatically
The next step in the evolution is the invasive radial growth phase, when individual cells
start to acquire invasive potential, thereby leading to cancer spreading.
The following step in the process is the invasive melanoma called the vertical growth
phase (VGP). The tumour attains invasive potential so it can grow into the surrounding
tissue and can spread into distant tissues through blood or lymph vessels. The tumour
thickness is usually more than 1 mm, and the tumour involves the deeper parts of the
dermis (Fig. 6).
During the VGP phase, the host elicits an immunological reaction against the tumour,
which can be visible by the presence and activity of tumour infiltrating
lymphocytes (TILs). These cells sometimes completely destroy the primary tumour and
this phase is called regression, which is the latest stage of the melanoma development.
24
In certain cases, the primary tumour is completely destroyed and only the metastatic
tumour is discovered.
Incidence
Many studies have reported increasing incidence rates of cutaneous melanoma during
the last fifty years and this rapid increase in incidence has led to the concept of
“epidemic melanoma”, thereby becoming a real sanitary emergency. The most affected
populations are represented by Australians (40/100.000 inhabitants) and populations of
New Zealand and Northern Europe. Also in Italy melanoma incidence has been rising 5-
7% every year, with an incidence of 9,97/100.000 for men and 8,24/100.000 for women.
Mortality
Every year almost 41.000 deaths for cancer worldwide are related to skin melanoma and
1400 of them occur in Italy. A white male living in industialized countries has 1/100
possibility to develop skin melanoma during his life. In addition, in the last decades
melanoma shows higher mortality among young and middle-aged people. In particular,
in the past five years, in Italy the number of deaths clearly related to skin melanoma
were 3178 in males and 2807 in females, with almost a doubled mortality incidence in
northern regions compared to southern ones.
Fig. 6 Malignant progression of cutaneous melanoma.
25
Prevalence
The prevalence proportion is the proportion of people found to have the condition with
the total number of people studied and it is correlated to both frequency and prognosis
of pathology. In italy the prevalence of skin melanoma is 48,4/100.000 inhabitants for
women and 101,6/100.000 inhabitants for men. The discrepancy between the sexes is
reale to the highest incidence and better survival for women. Inside the country,
prevalence is three-fold higher in Veneto and Emilia if compared with southern regions.
Risk factors
Both endogenous and exogenous risk factors for skin melanoma have been identified.
Endogenous risk factors
1. Number of naevi: number of naevi is the most important susceptibility factor,
independent from naevi dimension and distribution. In fact, skin melanoma
develops from a congenital or acquired naevus in the 22-57% of all cases. In
addition, the number of naevi is related to major risk of developing melanoma
from healthy skin.
2. Presence of atipical/displastic naevi: a single displastic naevus is correlated to a
two-fold higher risk to develope melanoma, while presence of ten or more naevi
results in a twelve-fold higher risk.
3. Familiarity: frequence of familial malignant melanoma varies from 5 to 10% of
total cases. Subjects with close-related parents (parents, brothers) affected by
melanoma are at risk of developing maligannt melanoma. The risk augments
with the rising number of related affected.
4. Previous melanoma: almoste 11% of subjects with melanoma develope a new
melanoma in the following five years. In addition, other melanomas can show
up during the lifetime.
5. Adulthood: melanoma in children and adolescents is very rare, even if its
frequency is rising in the past decades. During adulthood the frequency rises
rapidly.
Other risk factors are presence of various solar lentigos, fair and red-headed people and
individuals with blistering or peeling sunburns. Different phototypes seem to modulate
the cancerogenic effects of ultraviolet rays (UV).
26
Exogenous risk factors
Development of skin melanoma seem to be related to intense but intermittent sun
exposure, specially in usually non photo-exposed cutaneous areas. In addition, exposure
and sunburn during childhood becomes a more important risk factor.
The UV rays are the leading cause of melanoma (Iarc 1997). It has been demonstrated
that this type of cancer is more common in decreasing latitudes and rising altitudes. In
particular, UV-B rays are the more carcinogenic to: 1) direct damage to DNA, 2)
damage to mechanisms of DNA repair, 3) partial suppression of cell-mediated
immunity. UV-A rays, once considered harmless, increase the effect of UV-B and act as
co-carcinogenic agents. The risks arising from a history of sunburn are significant for
both those experienced in childhood, adolescence, or any age (Elwood and Jopson
1997).
As for the ionizing radiation, the onset of melanoma depends on the total dose
accumulated and time of accumulation (radiologists, airline pilots, radio-treated
patients).
Clinical aspects
Signs and symptoms
Early signs of melanoma are changes to the shape or color of existing moles or, in the
case of nodular melanoma, the appearance of a new mole on the skin. At later stages,
the mole may ulcerate or bleed (Fiddler 1995). Early signs of melanoma are
summarized by the mnemonic "ABCDE" staging (Friedman et al., 1985):
Asymmetry
Borders (irregular)
Color (variegated)
Diameter (greater than 6 mm)
Evolving over time
These classifications do not, however, apply to the most dangerous form of melanoma,
nodular melanoma, which has its own classifications:
Elevated above the skin surface
Firm to the touch
Growing
27
Metastatic melanoma may cause nonspecific paraneoplastic symptoms, including loss
of appetite, nausea, vomiting and fatigue. Metastasis of early melanoma is possible, but
relatively rare: less than 1/5 of early diagnosed melanomas become metastatic and brain
metastatsis represent the most typical result of metastatic melanoma. It can also spread
to the liver, bones, abdomen or distant lymph nodes. A recent new method of melanoma
detection is called the "ugly duckling sign" and it is commonly used because it is a
simple diagnostic procedure and it is highly highly effective in detecting melanoma.
Simply, correlation of common characteristics of a person's skin lesion is made. Lesions
which greatly deviate from the common characteristics are labeled as an "Ugly
Duckling", and further professional exam is required (Mascaro et al., 1998) (Fig. 7).
Fig. 7 Pictures and main characteristics of normali naevi and malignant melanoma.
Benign Malignant
Simmetrical
Asimmatrical
Regular shape Irregular shape
Homogenous
color
Variegated color
Smaller than 6
mm
Diameter greater
than 6 mm
Common
naevus
Changements in
dimension, shape
and/or other traits
28
Classification
Microscopically melanoma appears as a not-limited, asymmetrical lesion, consisting of
proliferation of melanocytes with atypical cytological characteristics, with a tendency to
migrate to the more superficial layers of the epidermis. Melanoma in situ, which is
located only into the epidermis and in the epithelium of skin appendages, can be
distinguished as follows: lentigo maligna type, superficial spreading type, acral
lentiginous type, uveal melanoma and nodular type (James et al., 2006).
Lentigo maligna melanoma is a melanoma that has evolved from a lentigo
maligna. It is usually found as a darkly pigmented raised papule, arising from a
patch of irregularly pigmented flat brown to dark brown lesion of sun exposed
skin of the face or arms in an elderly patient. Lentigo maligna is the non-
invasive skin growth and it is often considered as a melanoma-in-situ or a
precursor to melanomas. Once a lentigo maligna becomes a lentigo maligna
melanoma, it is considered and treated as if it were an invasive melanoma.
Superficial spreading melanoma, also known as "Superficially spreading
melanoma (SSM)”, is usually characterized as the most common form of
cutaneous melanoma in Caucasians (60-70% of total melanomas). The average
age at diagnosis is in the fifth decade, and it tends to occur on sun-exposed skin,
especially on the backs of males and lower limbs of females. This disease
commonly evolves from a precursor lesion, usually a dysplastic nevus, but I can
also arise from previously healthy skin. A prolonged radial growth phase, where
the lesion remains thin, may eventually be followed by a vertical growth phase
where the lesion becomes thick and nodular. As the risk of spread varies with
the thickness, early SSM is more frequently cured than late nodular melanoma.
Acral lentiginous melanoma is a kind of lentiginous skin cancer. Acral
lentiginous melanoma is observed on tpalms, soles, under the nails and in
the oral mucosa. Unlike the commonest forms of melanoma, acral lentiginous
melanoma does not seem to be directly connected to sun exposure. In fact, it can
occur on non hair-bearing surfaces of the body which may or may not be
exposed to sunlight, but is also found on mucous membranes. It is the most
common form of melanoma diagnosed amongst Asian and Black ethnic groups.
The average age at diagnosis is between sixty and seventy years. However, the
melanoma can also occur in Caucasians and in young people. Typical signs of
29
acral lentiginous melanoma include longitudinal tan, black, or brown streak on a
finger or toe nail, pigmentation of proximal nail fold and areas of dark
pigmentation on palms of hands or soles of feet.
Uveal melanoma is a cancer of the eye involving iris, ciliary body, or choroid,
collectively referred to as the uvea and tumours start from the melanocytes that
reside in the uvea and that are responsible for the color of the eye. These
melanocytes are distinct from the retinal pigment epithelium cells underlying
the retina that do not form melanomas. Several clinical and pathological
prognostic factors have been identified that are associated with higher risk of
metastasis of uveal melanomas. These include large tumour size, ciliary body
involvement, presence of orange pigment overlying the tumour, and older
patient age. Several histological and cytological factors are associated with
higher risk of metastasis, thereby including presence/extent of cells
with epithelioid morphology, looping extracellular matrix patterns, increased
infiltration of several types of immune cells, as well as staining with several
immunohistochemical markers.
Nodular melanoma (NM) is the most aggressive form of melanoma and grows
more rapidly in thickness than in diameter. Instead of arising from a pre-existing
mole, it may appear in a spot where a lesion did not previously exist. Because of
these charactyeristics the prognosis is often worse because it takes longer for a
person to be aware of the synptomes. NM is most often darkly pigmented, even
if some NM lesions can be light brown, multicolored or even colorless (non-
pigmented). A light-colored or non-pigmented NM lesion may escape detection
because the appearance is not alarming, however an ulcerated and/or bleeding
lesion is common. A particular variant of nodular melanoma, of viral origin, is
repreented by the polypoid melanoma. The microscopic hallmarks are dome-
shaped at low power, thin or normal epidermis, dermal nodule of melanocytes
with a 'pushing' growth pattern and no "radial growth phase".
Histopathological factors and prognosis
Features that are evaluated as affecting prognosis are tumour thickness in millimeters
(Breslow's depth), depth related to skin structures (Clark’s levels), type of melanoma,
presence of ulceration, presence of lymphatic/perineural invasion, presence of tumour-
30
infiltrating lymphocytes, location of lesion, presence of satellite lesions, and presence of
regional or distant metastasis (Day et al, 1981). Certain types of melanoma have worse
prognoses, but this is explained by their thickness. Interestingly, less invasive
melanomas, even with lymphnode metastases, carry a better prognosis than deep
melanomas without regional metastasis at time of staging. Local recurrences tend to
behave similarly to a primary unless they are at the site of a wide local excision, since
these recurrences tend to indicate lymphatic invasion. When melanomas have spread to
the lymphnodes, one of the most important factors is the number of nodes with
malignancy. Extent of malignancy within a node is also important; micrometastases in
which malignancy is only microscopic have a more favorable prognosis than
macrometastases. Macrometastases in which malignancy is clinically apparent as the
cancer completely replaces a node, have a far worse prognosis, and if nodes are matted
or if there is extracapsular extension, the prognosis is progressively worse. When distant
metastasis are detected, the cancer is generally considered incurable. The five year
survival rate is less than 10% and the median survival is 6 to 12 months. Metastases to
skin and lungs have a better prognosis, while metastases to brain, bone and liver are
associated with a worse prognosis.
Clark's level is a related staging system, used with Breslow's depth, which describes the
level of anatomical invasion of the melanoma in the skin (Balch et al., 2001) (Fig. 8).
Five anatomical levels are recognized, and higher levels have worsening prognostic
implications. These levels are:
Level 1 : Melanoma confined to the epidermis (melanoma in situ)
Level 2 : Invasion into the papillary dermis
Level 3 : Invasion to the junction of the papillary and reticular dermis
Level 4 : Invasion into the reticular dermis
Level 5 : Invasion into the subcutaneous fat
31
Fig. 8 Clark’s levels.
Medical treatment
Surgical treatment is the therapy of choice for the primary melanoma. The surgical
procedure involves the removal of the primary tumour with part of surrounding healthy
skin, reaching the muscle tissue. Currently, for patients with cutaneous melanoma
thicker than or equal to 1 mm, in combination with surgical excision of the primary
lesion, the sentinel limph node biopsy is performed, which is proved to be highly
sensitive in identifying clinically occult lymph node metastases. Therapy for metastatic
melanoma includes surgical radicalization of the lesion, use of chemotherapy or
combined protocols, or the use of radiation therapy for head can not be reached by the
drugs. Currently therapies are being tested with so-called "vaccines" which exploit the
biological response of the individual appropriately stimulated to slow the progression of
the disease.
Biological and genetic aspects
Melanoma is usually caused by DNA damage from UV light from the sun, but UV light
from sunbedscan also contributes to the disease.
A number of rare mutations, which often run in families, are known to greatly increase
one’s susceptibility to melanoma. Several different genes have been identified as
increasing the risk of developing melanoma. Some rare genes have a relatively high risk
of causing melanoma; some more common genes, such as a gene called MC1R that
32
causes red hair, have a relatively low risk.Genetic testing can be used to determine
whether a person has one of the currently known mutations.
One class of mutations affects gene CDKN2A. An alternative reading frame mutation in
this gene leads to the destabilization of p53, which is involved in apoptosis and its
mutations are detected in over the 50% of human cancers. Another mutation in the same
gene results in a loss of function of the inhibitor of CDK4, a cyclin-dependent kinase
that promotes cell division. Mutations that cause the skin condition xeroderma
pigmentosum (XP) also seriously predispose one to melanoma. Scattered throughout the
genome, these mutations reduce a cell’s ability to repair DNA. Both CDKN2A and XP
mutations are highly penetrant, meaning that the chances of a person carrying the
mutation to express the phenotype is very high (Golstein e Tucker, 2005).
Familial melanoma is genetically heterogeneous,and loci for familial melanoma have
been identified on the chromosome arms 1p, 9p and 12q. Multiple genetic events have
been related to the pathogenesis of melanoma. The multiple tumour suppressor 1
(CDKN2A/MTS1) gene encodes p16INK4a, low-molecular weight protein inhibitor
of cyclin-dependent protein kinases (CDKs), which has been localised to the p21 region
of human chromosome 9.
Other mutations confer lower risk, but are more prevalent in the population. People with
mutations in the melanocortin receptor 1 (MC1-R) gene, for example, are two to four
times more likely to develop melanoma than those with two wild-type copies of the
gene. MC1-R mutations are very common; in fact, all people with red hair have a
mutated copy of the gene. Also mutation of the MDM-2 SNP309 gene is associated
with increased risk of melanoma in younger women.
Other mutations affect genes regulating cell proliferation like Neuroblastoma RAS viral
(v-ras) oncogene homolog (N-ras), V-raf murine sarcoma viral oncogene homolog B1
(BRAF) and Phosphatase and Tensin homolog (PTEN) genes Pathogenically N-ras and
BRAF mutations seem to occur in early cancer development, while alterations in PTEN
gene are related to a late-stage development (Davies et al., 2002; Curtin et al., 2005;
Haluska et al., 2006).
33
STRESS HORMONES: EPINEPHRINE AND NOREPINEPHRINE
Definition of stress
Stress is commonly defined as the general reaction of the organism in order to respond
to endogenous and exogenous stimuli that can alter or affect the homeostasis of the
organism itself. The term stress has been first introduced in medicine by Hans Selye in
1936 (Selye 1936). Selye began to study on stress while trying to isolate sex hormones
in animals. By inoculating some toxic compounds in guinea pigs he could observe that
in animals occurred a common, almost “syndromic” reaction characterized by i) adrenal
hypertrophy , ii) atrophy of the thymus and lymph glands , iii) gastric ulcerations. He
further noticed that the same biological reaction, characterized by the common state of
hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis (see next paragraph),
it appeared in all the experimental animals regardless of the type of stimulus, called
"stressors". Selye defined the set of systemic reactions of the body resulting from
prolonged exposure to stressors as "general adaptation syndrome" (GAS). During the
following years, many and more complex research on the "new" concept of stress
started. In 1975, Mason demonstrated through research conducted on monkeys and then
humans that adrenocortical activation was not merely the result of exposure to the
stressor itself, but was specifically triggered by emotional reaction to stimuli. The
emotional arousal was characterized by the activation of the structures of the limbic
system (SL) that, as confirmed by following studies, represents the place of
coordination and control of stress reactions. The structures of the SL project abundantly
to the hypothalamic-pituitary axis. Therefore, the emotional arousal that is induced by
the stimulus is processed at the level of SL and occurs both at a biological/somatic
level, with autonomic and endocrine changes, and at a psychological/behavioral level
with sequences of motor behavior collectively known as the "flight or fight response"
(Mason 1975).
In particular Selye identified three basic phases: alarm reaction phase, resistance or
adaptation phase, and exhaustion, which follow one another in the body during each
stress reaction and collectively called "general adaptation syndrome" (GAS). GAS
syndrome is therefore a defense mechanism by which the body struggles to overcome
the difficulties to return as soon as possible to its normal operating balance.
34
The adrenal gland
The adrenal glands are equal endocrine glands, pyramid-shaped ( about 2 × 6 × 1 cm,
weight 4-6 g), located in the retroperitoneum superior to the kidneys, from which are
separated by an adipose capsule and renal fascia. Each adrenal gland is composed of
two structurally and functionally different parts, the adrenal cortex and the inner
medulla where the latter is localized internally and is completely surrounded by the
cortex. While the medullary part is nerve-derived, the cortex is organized in cellular
wrapped cords (zona glomerulosa), then assume a radial course toward the medulla
(zona fasciculata) and finally come together to form a coarse mesh (zona reticularis)
(fig. 9).
The outermost layer, the zona glomerulosa, is the main site for production of
mineralocorticoids, mainly aldosterone, which is largely responsible for the long-term
regulation of blood pressure. Aldosterone's effects are on the distal convoluted
tubule and collecting duct of the kidney where it causes increased reabsorption of
sodium and increased excretion of both potassium by principal cells and hydrogen ions
by intercalated cells of the collecting duct. Sodium retention is also a response of the
distal colon, and sweat glands to aldosterone receptor stimulation. The major stimulus
to produce aldosterone is represented by angiotensin II. Angiotensin II is stimulated by
the juxtaglomerular cells when renal blood pressure drops below 90 mmHg.
Situated between the glomerulosa and reticularis, the zona fasciculata is responsible for
producing glucocorticoids such as 11-deoxycorticosterone, corticosterone, and cortisol.
Cortisol is the main glucocorticoid under normal conditions and its actions include
mobilization of fats, proteins, and carbohydrates, but it does not increase under
starvation conditions. Additionally, cortisol enhances the activity of other hormones
including glucagon and catecholamines. Several factors, included emotional and
physical stressors, influence production of cortisol in response to adrenocorticotropic
hormone (ACTH) from the anterior pituitary gland. ACTH is secreted from corticotrope
cells expressed within the anterior lobe of the pituitary gland in response to the
corticotropin-releasing hormone (CRH), released by the hypothalamus. ACTH acts by
binding to cell surface ACTH receptors, which are located primarily on adrenocortical
cells of the adrenal cortex, thereby influencing steroid hormone secretion by both rapid
short-term mechanisms that take place within minutes and slower long-term actions.
In order to regulate the secretion of ACTH, many substances secreted within this axis
exhibit both slow/intermediate and fast feedback-loop activities. According to that,
35
glucocorticoids secreted from the adrenal cortex work to inhibit CRH secretion by the
hypothalamus, which in turn decreases anterior pituitary secretion of ACTH.
The most inner cortical layer, the zona reticularis, produces androgens, mainly
dehydroepiandrosterone (DHEA), DEHA sulfate (DEHA-S) and androstenedione,
which is the molecular precursor to testosterone.
The adrenal medulla is the core of the adrenal gland, and is surrounded by the adrenal
cortex. The chromaffin cells of the medulla, named for their characteristic brown
staining with chromic acid salts, are the body's main source of the
circulating catecholamines (CA). In particular, they secrete approximately 20%
norepinephrine (NE) and 80% epinephrine (E). To carry out its part of this response, the
adrenal medulla receives input from the sympathetic nervous
system through preganglionic fibers originating in the thoracic spinal cord from T5–
T11. Because it is innervated by preganglionic nerve fibers, the adrenal medulla can be
considered as a specialized sympathetic ganglion. Unlike other sympathetic ganglia,
however, the adrenal medulla lacks distinct synapses and releases its secretions directly
into the blood. Cortisol also promotes CA synthesis in the medulla. Produced in the
cortex, cortisol reaches the adrenal medulla and, at high levels, the hormone can
promote the upregulation of enzyme phenylethanolamine-N-methyltransferase (PNMT),
thereby increasing CA synthesis and secretion (Bertagna 2006; Cotran et al. 1999;
Cotran et al. 2000).
Fig. 9 Anatomic structure of the adrenal gland and hormones produced.
36
The periferic nervous system and the hypotalamic-pituitary-adrenal axis (HPA)
All vertebrates keep the correct homeostasis of their physiological mechanisms by
constant interaction between two main control systems, the endocrine and the nervous
system. The latter is anatomically divided into the central nervous system, which
includes the brain and the spinal cord, and the peripheral nervous system, which
comprehends all the nerves responsible for transmitting signals between the central
nervous system and the body’s glands, muscles and organs, collectively known as
effector organs.
The peripheral nervous system is organized in 43 pairs of nerves divided into 12 pairs of
cranial nerves and 31 pairs of spinal nerves. Each nerve is formed by nerve fibres
including the axons of efferent and/or afferent neurons. Therefore, the peripheral
nervous system is further divided into two main parts: the efferent division, which
coordinates signals going from the central nervous system towards effector organs, and
the afferent division, which transmits signals from peripheric receptors towards the
central nervous system.
The efferent division is further organized into the somatic nervous system, which
innervates voluntary skeleton muscles, and the autonomic nervous system, regulating
not-voluntary smooth and cardiac muscles, glands and gastrointestinal neurons.
Physiological and anatomical differences allow to divide the autonomic nervous system
into three further parts: sympathetic, parasympathetic and enteric. The sympathetic
nerve fibers originate from the central nervous system, particularly from the thoracic
and lumbar regions of the spinal cord, while parasympathetic nerves leave the central
nervous system in the brain and in the sacral region of the spinal cord.
Together with the nervous system, the endocrine system acts in order to integrate and
correctly coordinate the functions of the nervous system. Unlike the nervous system, the
endocrine system acts, in general, more slowly, normally in a span of time ranging from
30 minutes to three hours, and more diffusely, as all cells of the body can be reached by
hormonal signals, and primarily transimitting to peripheric organs and tissues.
In particular, the integration between these two systems is related to the HPA axis,
where hypotalamus is both part of the central nervous sytem and “interface” between
nervous system itself and the endocrine one. Subsequently, hypothalamus controls
periferic endocrine functions through pituitary gland stimulation (Kvetnansky et al.
2009) (fig. 10).
37
In the light of these observations, hypotalamus represents the most important gland for
elaboration of stress responses. In particular the paraventricular nucleus (PVN) is
considered as critical for generation of neurophysiological stress responses. The axons
of the parvocellular neurosecretory neurons of the PVN project to the median eminence,
a neurohemal organ at the base of the brain, where their neurosecretory nerve terminals
release their hormones. The median eminence contains fiber terminals from many
hypothalamic neuroendocrine neurons, secreting different neurotransmitters or
neuropeptides, including vasopressin (VPN), CRH, thyrotropin-releasing hormone
(TRH), gonadotropin-releasing hormone (GnRH), growth hormone-releasing hormone
(GHRH), dopamine (DA) and somatostatin into blood vessels in the hypothalamic-
pituitary portal system. In particular, CRH and VPN act synergistically in order to
stimulate ACTH secretion and glucocorticoids production from the adrenal cortex (Vale
et al. 1981). Several studies have in fact demonstrated that there is a positive, biunivocal
interaction between CRH and AVP where the secretion of one induces production of the
other and viceversa (Lamberts et al. 1984).
In absence of stressful conditions, both CRH and AVP are secreted every 2-3 hours into
the bloodstream following a pulsatile, circadian rythm (Engler et al. 1989). In resting
conditions these hormones are usually produced in early hours of the morning with
consequent rise of ACTH and cortisol circulating levels. This pulsatile secretion can be
disrupted during stressful events (Horrocks et al. 1990; Chrousos et al. 1998).
During a single, momentary stress there is a massive release of CRH and VPN, thereby
leading to a more frequent secretion of ACTH and cortisol. According to the type of
stress, other molecules like angiotensin II and various cytokines act as signaling
mediators inducing exponential activation of the HPA axis (Holmes et al. 1986; Phillips
1987).
In this light glucocorticoids represent the real final effectors of the HPA axis, thereby
directly contributing to regulation of body homeostasis and stress sistemic response. At
the same time they present a key role in regulating basal activity of HPA axis and in
blocking the sistemic stress response through a negative-feedback mechanism both on
hypotalamus and pituitary gland. According to this mechanism of action, the
stimulation of glucocorticoids on periferic organs is progressively reduced when the
stressful event is no longer present (De Kloet et al. 1991).
Glucocorticoids mediate their biological effects through activation of cytoplasmatic
receptors ubiquitously expressed. In their inactive form these receptors are complexed
38
with heat shock proteins (HSPs) that impair their binding to DNA. Following ligand
binding the receptor show a conformational change and dissociates from HSP. The new
receptor-ligand complex, in an homodimeric form, thereby migrates to the nucleaus
where it directly binds specific DNA sequences located in the target genes promoters
and named glucocorticoids responsive elements (GREs) (Smith et al. 1993; Pratt 1990).
The activated receptors also act through protein-protein interaction in order to regulate
specific signling pathways like the c-jun/c-fos and NF-kB mediated ones as positive
regulators of genes involve in activation and proliferation of immune system cells
(Scheinman et al. 1995)
Fig. 10 The hypotalamic-pituitary-adrenal axis (HPA).
Cortisol and other glucocorticoids hormones exert both anabolic effects in the liver and
anabolic effects in several peripheric tissues such as muscle, connectival, lymphoid and
adipose tissues. In particular, glucocorticoids effects are:
Inhibition of ACTH secretion. Cortisol inhibits ACTH secretion through a
feedback inhibition exerted both at the hypothalamus and at the pituitary levels.
In general all glucocorticoids inhibit ACTH secretion, and the more powerful is
the glucocorticoid action, the greater is the degree of inhibition.
Metabolic effects. Glucocorticoids stimulation increases liver glycogenolysis,
while decreasing glucose uptake in muscle, adipose and other tissues, thereby
inducing an increase in circulating glucose free levels. These actions synergize
39
in order to promote mobilization of energy sources, like amino acids, fatty acids
and glycerol, from some tissues to provide energy substrates, particularly
glucose, for others, notably the brain and heart, both characterized by high
glucose consumption rate. The metabolic effects of glucocorticoids may be
counterbalanced by those of other hormones, particularly insulin, the secretion
of which is in turn stimulated by the rise in glucose circulating levels.
Vascular reactivity. In addition to its effects on the organs and tissues directly
involved in metabolic homeostasis, cortisol influences various other organs and
systems. For example, cortisol maintains the responsiveness of vascular smooth
muscle to CA and therefore participates in blood pressure regulation. In adrenal
insufficiency, vascular smooth muscle becomes unresponsive to CA. The
decreased responsiveness, together with the associated hypovolemia caused by
mineralocorticoid deficiency, can result in severe hypotension (Chrousos 2007;
Taché et al. 2007).
Effects on central nervous system. Cortisol can directly modulate electrical
activity of the neurons via type I and type II glucocorticoid receptors that are
primarily expressed in limbic system and hippocampus. The ability of cortisol to
decrease hippocampal volume as well as memory has also been demonstrated.
Cortisol decreases REM sleep but increases both slow-wave sleep and time
spent awake. Excessive concentrations of cortisol in blood can cause insomnia
and strikingly increase or decrease mood. In addition, loss of sleep seems to be
correlated to elevated circulating levels of IL-6 despite the reduced stimulation
of IL-6 production exerted by catecholamines. This effect is probably related to
reduced glucocorticoids-mediated inhibition (Vgontzas et al. 1999; Vgontzas et
al. 2003).
Effects on immune system and inflammatory responses. Various studies have
demonstrated that cytokines and other umoral mediators of inflammatory
response act as powerful activators of stress response from the central nervous
system, thereby creating a molecular communication system between central
nervous system and immune system. For example three of the most important
proinflammatory cytokines, tumour necrosis factor α (TNF-α), interleukin-1b
(IL-1b) and interleukin-6 (IL-6) directly stimulate HPA axis activity during
cronic inflammatory diseases, both alone and/or through synergic actions
(Chrousos 1995; Tsigos et al. 1997). Some of these positive effects are indirectly
40
related to stimulation of central and peripheric catecholaminergic signaling
pathways. In particular activation of nociceptive, somatosensory and visceral
peripheric nerve fibres induce secretion of CRH and CA through spinal
ascendent fibres (Chrousos 1995), thereby leading to a strong impairement of
immune and inflammatory response as all cellular components are inhibited by
glucocorticoids production. These hormones decrease differentiation and
proliferation of local mast cells, stabilizes lysosomes and decreases production
of platelet activating factor (PAF) and nitric oxide (NO). Glucocorticoids also
suppress immune response by decreasing the number of circulating T-
lymphocytes and by decreasing the production of interleukins and interferon-γ
(IFN-γ) that are critical mediators of immune response (Chrousos 1995; Elenkov
et al 1999). In addition, cortisol and all known glucocorticoids suppress
synthesis and decrease the release of arachnidonic acid, the key precursor for a
number of mediators of inflammation like prostaglandins (PGDs) and
leucotrienes (LTs).
Effects on stress. ACTH and cortisol secretion are increased by stressful stimuli
including surgery, trauma, pain, infection, hypoglycemia and hemorrhage,
thereby inducing an higher secretion of NE and E from adrenal medulla. In
particular, locus ceruleus (LC) and other groups of cells secreting NE,
collectively known as the LC/NE system, contribute in increasing autonomous
and neuroendocrine stress responses through HPA axis activation. These
observations show a biunivocal connection between CRH production and
LC/NE system where CRH stimulate secretion of NE and viceversa (Taché
2007).
41
Biosinthesis of catecholamines
CA have been first characterized at the beginning of the twentieth century. CA are
synthesized from the amino acid precursor L-tyrosine (Fig. 11). There are two primary
sources of tyrosine, from the diet and from hydroxylation of the amino acid
phenylalanine in the liver.
Upon entry into an adrenal chromaffin cell, sympathetic or brain catecholaminergic
nerve terminals, tyrosine is converted to dihydroxyphenylalanine (DOPA) by the
soluble cytoplasmic enzyme tyrosine hydroxylase (TH). TH is an iron-containing,
biopterin-dependent aminoacid hydroxylase. It utilizes tyrosine, tetrahydrobiopterin
(BH4), and molecular oxygen to generate DOPA, dihydrobiopterin, and water. The
cofactor BH4 is resynthesized from dihydropterin by the enzyme dihydropteridine
reductase. Since BH4 is present in subsaturating levels, TH activity depends on its
availability. In humans, several isoforms of TH can arise from alternative splicing of a
single primary transcript. Primates exhibit only two of these isoforms, and lower
animals have only one. The significance of these isoforms, the physiology of the
alternative splicing, and whether it is affected by stress is not clear (Nagatsu 1991). TH
activity is intricately regulated in the short and long term.
In the short term, TH enzymatic activity is regulated by feedback inhibition; thus TH is
inhibited by catecholamines secretion (DOPA, NE, DA). TH is also regulated by
allosteric regulation and direct protein phosphorylation. TH can be phosphorylated by a
variety of kinases at several serines (positions 8, 19, 31, and 40) in the NH2-terminal
domain, but the role of this post-transcriptional modification has not been clarified yet.
On the other hand, in the medium to long term, TH can be regulated by enzyme
stability, transcriptional regulation, RNA stability, alternative RNA splicing, and
translational activity. Change in TH gene expression is a major mechanism whereby the
catecholaminergic system responds to stress. For example, CREB is one of the
mediators of the transcriptional response to stress: CREB is one of the transcription
factors of the CREB/ ATF family that then binds as dimers to the cAMP response
element (CRE) on a variety of genes, including TH. CREB phosphorylation on serine-
133, necessary for its pro-transcriptional activation, is found elevated in the LC during
exposure to single and repeated stressful events and this leads to increased TH
expression (Nagatsu 1995).
Subsequently DOPA is converted into DA by a nonspecific enzyme, aromatic L-amino
acid decarboxylase (AAAD) whose activity strongly depends upon availability of its
42
cofactor, pyridoxal phosphate. The most high DA-producing region in the nervous
system is represented by the corpus striatum, which receives the projections from the
substantia nigra and has a central role in regulating motor coordination. For example, in
Parkinson’s disease loss of dopaminergic neurons inside the substantia nigra lead to its
peculiar motor disfunction.
Dopamine is then taken up from the cytoplasm into storage vesicles and converted into
NE by dopamine hydroxylase (DBH), an enzyme found in soluble and membrane-
bound forms within storage vesicles.
Both forms are encoded by the same mRNA. DBH activity utilizes Cu2+
, ascorbic acid,
and O2. The NE is mainly synthesized in the adrenal medulla and the central and
peripheral nervous system, where it is used by cells in the ganglia of the sympathetic
nervous system, thereby showing that NE represents the main neurotransmitter in this
section of the peripheral autonomic nervous system. It is also used in the locus
coeruleus where it influences states of sleep and wakefulness, attention, and eating
behavior.
NE is then converted into E by the soluble cytoplasmic enzyme phenylethanolamine N-
methyltransferase (PNMT) that uses S-adenosyl-methionine as the cofactor and whose
activity is inducible by glucocorticoids. PNMT is mainly localized in the adrenal
medulla; however, sympathetically innervated organs and some brain areas are also able
to synthesize small amounts of E. Separate populations of adrenal chromaffin cells
contain NE and E as the final products of CA biosynthesis. PNMT gene expression was
found also in some nonneuronal cells in the heart and skin (Ziegler et al. 1998).
43
Fig. 11 Biosinthesis of catecholamines.
Release and action of catecholamines
The process of CA release is similar in the adrenal medulla and the sympathetic nerve
endings (fig. 12). Acetylcholine released from sympathetic preganglionic nerve
terminals binds to nicotinic cholinergic receptors and leads to a depolarization of cell
membrane, resulting in an increase in membrane permeability to sodium. This initiates a
series of events that lead to an increase in the influx of calcium. Then, CA storage
vescicles fuse with the chromaffin or sympathetic neuronal cell membrane and release
via exocytosis their contents of CA, together with chromogranins, other neuropeptides,
ATP and a fraction of the soluble DBH, via exocytosis. However, the exact mechanism
of Ca2+
evoked exocytosis is not clear (Young et al., 1998).
Currently, more than 30 biologically active substances have been localized in adrenal
chromaffin, sympathetic neuronal, and brain catecholaminergic cells, and a number of
44
them are released following depolarization of the cell membrane. In addition, various
biologically active neuropeptides are colocalized with acetylcholine within sympathetic
preganglionic nerve terminals like substance P, neuropeptide Y (NPY) and vasoactive
intestinal polypeptide (VIP), and they all appear to act as neuromodulators or
cotransmitters of cholinergic transmission (Young et al., 1998; De Diego et al., 2008).
In particular, the VIP peptide has a key role in stimulating catecholamines release, while
substance P modulates acetylcholine secretion. Finally, the neuropeptide NPY seems to
also exert a trofic effect on sympathetic nerve terminations and has been recently
described as the major factor responsible for stress-induced obesity (Zukovska et al.,
1998; Kuo et al., 2007; Kuo et al., 2008). Another example is represented by protein
sinexin, also named annexin VII, which exerts a role in mediating the signaling pathway
responsible for vescicles fusion to neuronal cell membranes, while other proteins such
as cathestatin have an inhibitory effect on catecholamines release (Pollard et al., 1998;
Kennedy et al., 1998).
After release in the intersynaptic space, CA-mediated biological effects are quickly
blocked through two main processes:
Conversion of CA in biologically inactive products through enzymatic action of
monoammine oxidase (MAO) in neuronal cells and catechol-O-
methyltransferase (COMT) in non-neural cells.
Re-uptake of active CA and formation of new cytoplasmic storage vescicules in
both sympathetic and effectors neural cells.
MAO is a mitochondrial flavoprotein located in the outer membrane of presynaptic
neurons that catalyzes deamination of amines, with production of aldehydes that are
metabolized to carboxylic acids or alcohols. MAO-A subtype has a higher affinity for
NE and E and is highly localized in brain neurons. MAO-B is responsible for
degradation of DA. In sympathetic nerves, the aldehyde produced from NE and, to a
less extent, from E by MAO-A is converted to dihydroxyphenylglycol (DHPG).
Subsequent extraneuronal O-methylation leads to production of 3-methoxy-4-
hydroxyphenylglycol (MHPG).
COMT is involved in the inactivation of the catecholamines through addition of a
methyl group to the catecholamine, which is donated by S-adenosyl methionine. In
particular, the enzyme catalyzes reactions of methylation of NE in normetanephrine and
of E in metanephrine. COMT can also be found extracellularly, although extracellular
45
COMT plays a less significant role in the CNS than it does peripherally where is
primarily expressed in the liver (Golan and Armen).
The mechanisms of neural uptake act synergistically with COMT and MAO enzymatic
activity. In particular, neuronally released CA are inactivated by “uptake 1” (U1) in
combination with enzymatic degradation by MAO, while circulating CA are inactivated
by “uptake 2” (U2) and COMT-mediated enzymatic degradation (Axelrod et al., 1957;
Axelrod et al., 1959).
Uptake 1 (U1) acts in order to recapture locally released NE or circulating NE to save it
by intraneuronal storage for reuse. U1 requires energy and is a mechanism carrier-
mediated, where the carrier involved moves CA against large concentration gradients.
Since U1 acts as a first-order kinetic process, U1 increases in parallel with increase NE
release during exposure to stressors. In fact, about 90% of released NE is reuptaken to
neurons through two main transporters, NE transporter (NET) and dopamine transporter
(DAT), while it seems to have a less important role in circulating E inactivation
(Eisenhofer et al., 1990; Eisenhofer et al., 1991). In particular, NE is translocated by
NET about twofold more effectively than E. This explains why sympathetic nerves take
up NE more efficiently than E. Dopamine is a much better substrate for DAT than NE
or E. NET and DAT-mediate transport is Na+ and temperature-dependent and is
characterized by high catecholamines affinity, but very low capacity. In brain, TH and
DAT are considered characteristic markers for dopaminergic neurons. While DAT is
mainly expressed in brain dopaminergic cells, neither NET nor DAT expression is
restricted to central or peripheral noradrenergic neurons. Uptake of NE by NET also
takes place in some extraneuronal cell types that express the same NET isoform that is
expressed in noradrenergic neurons. Extraneuronal sites of NET expression are in the
chromaffin cells of adrenal medulla, in lung and placental tissues, while extraneuronal
DAT expression was found in the gastrointestinal tract, pancreas and kidney
(Eisenhofer et al. 2001).
Extraneuronal uptake 2 (U2) is an active process of transport into non-neuronal cells.
The extraneuronal monoamine transporter of U2 (EMT) has little stereospecificity and
has very low affinity and specificity for CA. In particular, unlike U1, U2 favors E over
NE and is not a Na+ and Cl
- -dependent process. U2 is responsible for formation of CA
metabolites in liver, kidney, and lung and is highly sensitive to inhibition by
glucocorticoids. As a negative feedback control mechanism, U2 activity is strongly
46
impaired by CA O-methylated metabolites normetanephrine, metanephrine, and by
corticosteroids. (Bonish 1980).
In addition, until now at least three catecholamines transporters located in non-neural
sites have been described and named cationic organic transporters (OCT)-1, 2 e 3, all
carachterized by high specificity for DA transport (Eisenhofer et al. 2001).
Subsequently reuptaken CA are stored into new cytoplasmic vescicules through action
of the monoamin vescicular transporter (VMAT). Two isoforms of this tranporter have
been described until now: VMAT-1, considered as the “neuroendocrine form”, and
VMAT-2, also known as the “neuronal form”, strictly expressed in the central and
peripheric nervous systems. As for the molecular transporters previously described, also
VMATs transport molecules against concentration gradient thereby exploiting the
protonic gradient created by ionic H+ channels (Goldstein 2001).
In conclusion, fast enzymatic inactivation/reuptake of CA released into the synaptic
cleft is a prerequisite for fine control over the effector system. Contrary to the usual
depictions, vesicular stores of CA do not exist in a static state simply waiting for
exocytotic release. Rather, they exist in a highly dynamic equilibrium with the
surrounding cytoplasm, with passive outward leakage of CA, counterbalanced by
inward active transport under the control of VMAT (Eisenhofer et al. 2004).
In spite of the fact that this process is highly activated by stress, the studies on stress-
induced changes in activity and gene expression of CA transporters located in the
peripheral neurons are quite rare. Very few reports deal with NET transporter protein
and gene expression during oxidative stress in PC12 cells. These results support a
functional role of oxidative stress in mediating the neuronal NE uptake associated with
reductions in NE uptake binding sites and NET protein production, without changes in
NET gene expression. The effect of oxidative stress on NET is a post-transcriptional
event (Mao et al., 2004; Mao et al., 2005).
47
Fig. 12 Mechanisms of secretion, action and re-uptake of catecholamines in central and periferic
neuronal cells.
The adrenergic receptors
In order to mediate their biological effects, CA bind to specific receptors named
adrenergic receptors, all included in the superfamily of transmembrane seven domains
receptors coupled to G proteins. In 1948 Ahlquist first identified and classified these
receptors in two main types, alpha (α) and beta (β), according to their responsiveness to
in vitro stimulation with different classes of molecular agonists and antagonists
(Ahlquist 1948). Almost fifteen years later α receptors have been further divided into
two classes according to their anatomic location, thereby defining as α1 post-synaptic
receptors and as α2 the pre-synaptic ones (Langer 1974). Further biological and
pharmacological studies have led to the final classification of adrenergic receptors in
distinct subtypes. In particular, α1 receptors have been divided in α1a, α1b and α1d, while
α2 receptors have been classified in α2a, α2b and α2c adrenoceptors.
48
β receptors are also heterogenous and were initially subdivided into β1 and β2
adrenoceptors on the basis of functional and ligand affinity studies. Subsequently the β
receptors have been classified using functional studies, receptor binding and genetic
techniques. The β adrenoreceptor family is now divided into three disctinct subtypes,
the β1, β2 and the atypical β3 receptor. There is an additional β-adrenoceptor subtype
which has been recently identified in cardiac tissue and is a putative, atipical subtype
named β4 adrenoceptor (Lands et al. 1967; Strosberg e Pietri-Rouxel 1996; Kauman
1997) (fig. 13).
Fig. 13. α and β receptors classification.
α1: these receptors are located in the central and periferic nervous system. In
particular, in the central nervous system they are highly expressed post-
synaptically where they mediate an excitatory role. On the other side, peripheral
α1 receptors are predominantly located intrasinaptically on both vascular and
non-vascular smooth muscle where receptors activation results on muscle
contraction and increase of blood pressure. They are also located on the cardiac
muscle where they mediate a positive inotropic effect and on the liver, activating
glycogen phosphorylation. (Bylund 1992; Aboud et al. 1993).
All these receptors are coupled to phospholipase C and their activation induces
production of second messengers like inositole 3-phosphate (IP3) and
diacylglycerol (DAG). This signalling pathwat leads to activation of ionic
channels voltage-dependent and stimulation of protein kinase C (PKC) and
phospholipase A2, thereby regulating ciclic AMP (cAMP) and arachidonic acid
(AA) production (Harrison et al. 1991; Berridge e Irvine 1989).
49
α2: the heterogenous family of these receptors has been characterized studying
their different binding affinity eterogenic, thereby allowing the identification of
different receptors subtypes even within the same tissue. These receptors are
now divided in α2a, α2b, α2c e α2d receptors (MacKinnon et al. 1994). In
particular, the α2d receptor shows an unique pharamcological profile, but it is
still considered a splicing variant or receptor α2a for its high sequence homology
with this receptor (fig. 9) (Simonneaux et al. 1991).
α2 receptors are expressed both at a pre and post-synaptical level where they
mediate inibitory effects on the central and periferic nervous system (French
1991). In the central nervous system they regulate neurotransmitter release
through autoreceptors located on noradrenergic nerve terminals and
heteroreceptors located on other neurotransmitter signals. In particular, their
sedative properties, mediated by somatodendritic autoreceptors on the locus
coeruleus, have led to the development of α2-agonists as sedatives and
anesthetics. Other central effects of α2 adrenoceptors include the regulation of
blood pressure, pupil diameter and body temperature. Peripheral activities
include contraction of vascular smooth muscle and inhibition of lypolyisis
through activation of the receptors expressed by fat cells.
The activity of α2 receptors is mediated by the activation of different G proteins
including Gi/Go proteins. All the receptor subtypes are negatively coupled to
adinilate cyclase, thereby mediating an inhibitory effect on cAMP production
(fig. 14). In addition, it has been recently characterized a role of these receptors
in activating several types of ionic channels like K+ and Na
+/H
+ transporters
(Bylund et al. 1995).
β1: β1 receptors are located in high density in the striatum and a selective
decrease in the expression of these receptors is related to Huntington’s Corea
development (Waeber et al. 1991). β1 receptors are also expressed in kidneys,
cardiac muscle, airway muscle and fat cells and they show similar affinity for
both NE and E binding. It is an excitatory receptor and its activation leads to
positive cardiac inotropic and chronotropic effects, while in kidneys it stimulates
renin secretion from iuxtaglomerular cells.
β1, β2 and β3 receptors positively regulates adenilate cyclase through activation
of small Gs protein, thereby stimulating voltage-dependent Ca2+
ionic channels
(fig. 14) (Bylund et al. 1994).
50
β2: it is an inhibitory receptor and shows higher affinity for NE binding in
comparison with E. This receptor is mainly expressed on gastrointestinal and
airway smooth muscle cells and its activation results in bronchodilation and
general muscle relaxation. It is also located on blood vessels and coronaric
arterie, increasing organs perfusion.
β3: is an excitatory receptor and it is located mainly in adipose tissue, both white
and brown, where it is involved in the regulation of lipolysis and thermogenesis
by NE through activation of the enzyme lipase. For this reason β3 agonists are
good candidates for obesity treatment (Kauman 1997). Some other β3 agonists
have also demonstrated antistress effects in animal studies, suggesting it also has
a role in the central nervous system. β3 receptors are found in the gallbladder and
in urinary bladder. Their role in gallbladder physiology is unknown, but they are
thought to play a role in lipolysis and thermogenesis in brown fat. In the urinary
bladder it is thought to cause relaxation of the bladder and prevention of
urination.
Fig. 14 α and β adrenergic receptors and their signalling pathways.
51
Catecholamines and cancer
Over the past 25 years, epidemiological and clinical studies have linked psychological
factors such as stress, chronic depression, and lack of social support to the incidence
and progression of cancer. In particular, several molecular and animal studies have
begun to identify specific signaling pathways that could explain the impact of
neuroendocrine effects on all stages of tumour growth and metastasis, from proliferation
and growth of the primary tumour to metastatization of distant tissues (fig. 15) (Reiche
et al., 2004; Sood and Thaker, 2008).
Adhesion to ECM
The tumour cell ability to adhere to ECM represents a key event leading to
metastatization of distant organs. In particular, integrins receptors expressed on cells
membrane are key mediators for ECM-cell interaction, and recent studies have started
focusing on the role of CA in regulating cancer cells adhesion. Despite this mechanism
have not been fully elucidated yet, it is well known that cAMP has the ability to regulate
activity of the adhesion-mediating small GTPases RhoA and Rac through the activation
of protein kinase A (Mercurio and Rabinovitz, 2001). Recent studies also showed that
the exchange factor directly activated by cAMP (Epac) is also involved in integrin–
mediated cell adhesion and cell-cell junction formation. According to these
observations, recent data show that the β-agonist isoproterenol promotes ovarian cancer
cell spreading and adhesion to laminin-5 in an Epac-dependent way and promotes
adhesion to a fibronectin matrix in a cAMP mediated Epac-Rap1 pathway. Thus, stress
hormones may promote cancer cell-matrix attachments and this mechanism represent a
promising therapeutic target in order to avoid cancer cells migration from the primary
tumour to distant peritoneal sites (Bos, 2006).
Proliferation and growth of primary tumour and metastasis
At both primary and metastatic sites, activation of autocrine, paracrine and/or endocrine
pathways can promote tumour cell proliferation by disrupting the balance between
positive, pro-proliferative and inhibitory signals (Langley and Fidler, 2007). Until now,
data about the role of CA on cancer cells proliferation seem to be quite controversial
and closely related to the studied cell type. In fact, some observation suggest that
catecholamines repress normal cell proliferation, such as slowing keratinocyte growth,
resulting in reduced wound healing in the context of stress (Flaxman and Harper, 1975).
52
On the other hand, other studies show that β-adrenergic inhibition with β2 adrenergic
receptor agonist pirbuterol reduces human tumour cell growth in xenograft models
through inactivation of the Raf-1/Mek-1/Erk1/2 pathway (Carie and Sebti, 2007) and, in
breast cancer, some studies have directly related β2-adrenergic receptor activation to
increased tumour growth and progression. In other tumour models, CA inhibition on
cancer cells proliferation have been related to dopamine and α-adrenoceptors signaling.
For example, studies focused on a neuroblastoma model showed that NE inhibited
cancer cell growth in cells expressing the dopamine transporter, where treatment with
NE induced an increased ratio of cells undergoing G0/G1 phase (Pifl et al., 2001).
β2-adrenergic receptors may also act synergistically with other stimuli on cancer cells
proliferation. A recent study showed that in a gastric cancer model these receptors
contributeto nicotine-induced activation of the protein kinase C/Erk1/2/cyclooxygenase-
2 (COX-2) pathway, leading to enhanced tumour cells proliferation (Shin et al., 2007).
Another factor regulated by adrenergic signaling is represented by the transcriptional
factor cAMP responsive element binding (CREB) protein, which can be activated in
response to external stimuli such as stress hormones. It has been clearly demonstrated
that CREB plays an important role in tumour cell proliferation, migration, angiogenesis,
and apoptosis inhibition (Jean and Bar-Eli, 2000). Thus, β-adrenergic receptor signaling
might interact with other CREB activators in order to modulate molecular processes
involved in tumour progression, like viral infections. Infective events are key co-factors
in the initiation of approximately 20% of human tumours, and all major human tumour-
associated viruses have been found to be activated by either β-adrenergic or
glucocorticoid-mediated signaling pathways (Antoni et al., 2006). For example, human
herpesvirus 8, responsible for Kaposi’s sarcoma, activates a cAMP response element in
the promoter of a key viral transcription factor, thus β-adrenergic stimulation of the
viral host cell induces CREB-mediated expression of viral oncogenes and growth
factors that promote viral infection spreading and progressively lead to cancer initiation.
Also Epstein-Barr virus and high-risk variants of the human papilloma virus are
similarly activated by glucocorticoids action (Antoni et al., 2006).
Finally, in a prostate carcinoma model, treatment with cAMP agonists resulted in
epithelial prostate cancer cells acquiring neuroendocrine characteristics (Cox et al.,
1999). These characteristics were represented by dense core granules in the cytoplasm,
the formation of neuron-like processes, loss of mitogenic activity, and new expression
of neuroendocrine markers. The presence of these neuroendocrine-like cells has been
53
linked to poor prognosis in prostate cancer patients as they have minimal proliferative
ability, but promote proliferation of surrounding cells through paracrine stimulation
(Cohen et al., 1991).
Angiogenesis
Recently, it has been found that stress hormones may promote angiogenic mechanisms
in human tumours, thereby leading to increased growth both of the primary tumour and
metastasis. In adipose tissues, nasopharyngeal cancer cells and in two ovarian cancer
cell lines, VEGF has been shown to be upregulated by NE through activation of β-
adrenergic receptor signaling pathway cAMP/protein kinase A (PKA)-dependent,
(Lutgendorf et al., 2003; Yang et al., 2006). The effect of NE on VEGF stimulation was
completely reverted after treatment with non selective β-blocker propanolol and
mimicked by agonists of β-adrenergic receptors (Thaker et al., 2006). Furthermore, NE
modulates the expression of VEGF through activation of β2-adrenergic receptor in non-
solid tumours, such as multiple myeloma (Yang et al., 2008). Observations about the
effects of stress hormones on tumour angiogenesis have been confirmed both in vivo
and in vitro cancer models. In an ovarian cancer orthotopic model, chronic stress
induced by daily restraint resulted in higher levels of circulating and tissutal CA,
increased tumour burden, increased number of microvessels, and a more invasive
phenotype. Moreover, these same samples showed increased VEGF levels. According
to that, clinical data have demonstrated that there is an association between higher levels
of social support and lower serum VEGF levels in ovarian cancer patients (Lutgendorf
et al., 2002). Continuous infusion of a non-selective β-blocker partially abrogated the
effects of stress on tumour growth and progression, suggesting that β-adrenergic
receptors play an important role in stress-mediated tumour growth (Thaker et al., 2006).
Interleukin 6 (IL-6) is another key cytokine that plays a key role in tumour progression
and angiogenesis and, according to this observation, it is related to increasing
microvessel density and poorer cancer outcomes (Kiecolt-Glaser et al., 2003; Nilsson et
al., 2005). Lowering stressful conditions exerts a protective effect and correlation
between IL-6 with biobehavioral factors acts both at the primary tumour level and
distant organism sites (Costanzo et al., 2005). These clinical data have been confirmed
in vitro in an ovarian cancer model, showing that NE significantly increased IL-6
expression and that this increase was due to CA-mediated transcriptional regulation of
IL-6. Additionally, NE induced IL-6 production in ovarian carcinoma cells was
54
regulated by a β-adrenergic receptor/Src tyrosine kinase pathway (Nilsson et al., 2007).
The signal transducer and activator of transcription factor-3 (STAT-3) is activated by
growth factors and cytokines, such as IL-6 and VEGF and has been found to promote
angiogenesis and suppress apoptosis (Antoni et al., 2006). It is known that cytokines
such as IL-6 can contribute to tumour growth and progression through the activation of
STAT-3. Recent studies in an ovarian cancer model have determined that both NE and
E activate STAT-3, promoting its translocation to the nucleus and binding to DNA.
These effects on STAT-3 were mediated by β-adrenergic receptors and PKA signaling
and were independent of IL-6, thereby suggesting that stress-mediated tumour
progression may result, in part, through STAT-3 activation of downstream effector
pathways (Landen et al., 2007).
Migration/invasion
There are now several lines of evidence suggesting that stress hormones can promote
tumour cell movement and invasion in order to reach blood or lymphatic vessels that
facilitate tumour spreading. For example, in a breast cancer model, NE induced not only
a chemotactic response, but also promoted chemokinetic migration (Drell et al., 2003).
NE can also promote migration in a phospholipase Cγ and protein kinase Cα dependent
manner. In fact it has been demonstrated the role of CA and glucocorticoids on the
invasive potential of ovarian cancer cells, and MMPs that are important for tumour cell
penetration of extracellular matrix (Sood et al., 2006). NE concentrations similar to
those found in the bloodstream in stress conditions significantly increased the in vitro
invasiveness of ovarian cancer cells. E also promoted the invasive potential of ovarian
cancer cells and these effects were blocked by a β-adrenergic receptor antagonist.
Additionally, NE increased the in vitro production of MMP-2 and MMP-9 by tumour
cells, and pharmacologic inhibition of MMPs blocked NE mediated increase in tumour
cell invasion. Similar findings have been demonstrated in a nasopharyngeal carcinoma
model where catecholamines increased the invasive potential by increasing MMP-2 and
MMP-9 levels as well (Yang et al., 2006).
Resistance to apoptosis/anoikis
The continuation of the metastatic process depends on the ability of the tumour cell to
avoid apoptosis and anoikis (Langley and Fidler, 2007). It has been shown that
55
dopamine and NE can promote cellular apoptosis through a G protein-mediated
signaling in neuroblastoma cells (Chan et al., 2007). In addition, both acute and chronic
stress reduced the sensitivity of prostate and breast cancer cells to apoptosis through a
PKA-dependent BAD phosphorylation that was mediated by E interaction with β-
adrenergic receptors (Sastry et al., 2007). These in vitro data have been confirmed by
clinical trials showing a more positive outcome for patients suffering of prostate cancer
under daily treatment with β-blockers (Perron et al., 2004).
Furthermore, CA may also act together with glucocorticoids to promote cancer growth.
For example, cortisol increased the isoproterenol-induced cAMP and β-adrenergic
receptor accumulation on the cellular membrane and substantially increased the effects
of IL-1α, IL-1β, and TNF-α in lung carcinoma cells (Nakane et al., 1990). In addition,
synergistic action of CA together with glucocorticoids can counteract chemotherapy-
related cytotoxic effects in cervical and lung carcinoma cells through reduced
expression of pro-apoptotic factors involved in intrinsic apoptothic pathway (Herr et al.,
2003). According to these observations, breast cancer cell lines pre-treatment with
dexametasone impaired chemotherapy effects through glucocorticoid receptors
activation. In particular, glucocorticoids promote mitogen-activated protein kinase
phosphatase-1 (MKP-1) and glucocorticoid-inducible protein kinase-1 (SGK-1)-
mediated pathways. Glucocorticoids-mediated anti-apoptotic effects could be reversed
through MKP-1 and SGK-1 gene silencing (Wu et al., 2004).
Finally, anoikis is a cell process by which normal cells enter apoptosis when separated
from the extracellular matrix and neighboring cells. Recently, it has been demonstrated
that CA can protect ovarian cancer cells from anoikis. These effects are mediated by
focal adhesion kinase (FAK) phosphorylation through ADRB2-dependent activation of
Src kinase. Parallel results were observed in ovarian carcinoma patients, linking
increased levels of stress/depression to increased FAK activation and demonstrating
accelerated cancer progression in patients with high levels of FAK activity (Sood et al.,
2010).
56
Fig. 15 Effect of stress and related factors on cancer cells and tumour microenvironment.
57
HYPOXIA AND HYPOXIA INDUCIBLE FACTORS (HIFs)
Tumour microenvironmeent alterations like hypoxia, resulting from lack of correct
vascularization and/or excessive oxygen consumption (O2), can directly influence
cancer growth and progression through disruption of structrures and functional tissue
responses. All these evenbts contribute to resistance to pharmacological therapy and bad
prognosis. Major causes implicated in creation of hypoxic microenvironment are mostly
structural/functional alteration of tumour vessels and a growing distance between blood
vessels and cancer cells which strongly impairs O2 diffusion and delivery (Fig. 16).
Fig. 16 Tumoural hypoxia. Violet sreas represent hypoxic areas, while the grey ones represent the
necrotic areas.
Solid malignant tumours are usually characterized by elevated number of hypoxic
tissutal areas, with an O2 pression ≤ 2.5 mmHg, eterougenously located inside the
tumour burden. These regions may be located adjacent to regions with normal O2 partial
pressures (pO2) but, in contrast to normal tissue, neoplastic tissue can no longer fulfill
physiologic functions (Vaupel et al. 2001).
Hypoxia can be related to various factors dependent from perfusion, diffusion or anemic
conditions (Vaupel et al., 2001; Vaupel et al., 2002). Perfusion-correlated hypoxia is
caused by inadequate blood influx towards tissues. Tumoural microvessels often show
both structural and functional abnormalities like disorganized microcirculation,
perivascular detachment, vessels dilatation, irregular shape, loss of
physiological/pharmacological receptor and complete absence of blood flux regulation.
So new tumoural blood vessel formation is a relatively fragile process, subject to
58
disruptive interference at several levels and leads to frequent, but transient, ischemic
events.
Diffusion-related hypoxia is characterized by a progressively growing distance between
cancer cells and tumour microcirculation as a result of tumour mass growth. The final
effect is a continuosly reduced O2 delivery towards cells >70µm distant from blood
vessels, thereby leading to a condition of cronic hypoxia. Cronic hypoxia is also
correlated to changements in the “geometry” of O2 diffusion like modifications of blood
flux paths inside cancer vessels.
Finally, reduced ability of red blood cells to delivery O2 following anemic conditions
related either to therapy or tumour itself causes anemic hypoxia.
In conclusion hypoxia acts a continuative selective pressure by promoting growth and
proliferation of genetically alterated tumoural clones with diminished apoptotic
potential, thereby promoting cancer aggressiveness. The increasing inability of tumour
cells to activate apoptotic pathways can explain many of the clinical consequences of
malignant progression, such as locoregional and distant tumour propagation and
resistance to nonsurgical therapy. Survival and proliferation of occult perifocal tumour
cells with diminished apoptotic potential, located in hypoxic surgical scars, appear to be
major pathogenetic events in the formation of local recurrences (Graeberg et al. 1996;
Soengas et al. 1999).
Hypoxia is also responsible for accumulation of genomic mutations by cancer cells,
thereby increasing genomic instability favouring tumour progression and aggressiveness
(Bindra e Glazer. 2005; Bindra e Glazer 2007; Huang et al. 2007; Koshiji et al. 2005).
Hypoxic stress, both in vivo and in vitro, can generate DNA damage and mutations
(Møller et al. 2001). Hypoxia-induced DNA damage has been detected throughout the
bodies of individuals exercising at high altitude and has been attributed to ROS
produced upon reoxygenation (Risom et al. 2007) or to stress-induced leakage of ROS
from mitochondria (Møller et al. 2001). Also, severely hypoxic conditions and the
subsequent reoxygenation progressively decreases the activity of DNA repair
mechanisms (Bindra e Glazer. 2005). All these evidences have been confirmed through
in vitro and in vivo studies where esposition of cancer cells to hypoxia have been
demonstrated to actively promote their invasive and metastatic potential (Young et al.
1988; Young e Hill 1990; Cairns et al. 2001; Postovit et al. 2002; Rofstad et al. 2002).
Recent studies also suggest that sustained or intermittent hypoxic stress like cancer cells
exposition for 6-8 hours at pO2 ≤7 mmHg induces genetic alterations through both post-
59
transcriptional and post-translational modifications that modify cancer cells proteomic
profile (Vaupel et al 2001; Vaupel et al 2002; Höckel e Vaupel 2001). These
modifications include blockage of cell growth or damages to mechanisnìms of cell cycle
arrest, differentiation, apoptosis and necrosis (Moudler e Rockwell S. 1987; Durand
1991; Giacca 1996; Riva et al. 1998; Haroon et al. 2000). Physiologically hypoxia-
induced cell cycle arrest at G1/S checkpoint is related to hypoxia inducible factor-1α
(HIF-1α) which activates inhibitors of cycline-dependent kinases p21 e p27 (Goda et al.
2003). This mechanism of regulation seems to be p53-independent despite its
accumulation during hypoxic conditions (Koumenis et al. 2001). In fact, hypoxia-
induced apoptosis has been shown to be dependent on p53, Apaf 1, caspase 9, and
caspase 3, indicating that the mitochondrial apoptosis pathway plays a significant role in
this form of death, but hypoxia is also able to activate p53-indipendent apoptosis
through a Bcl-2-dependent mechanism (Soengas et al. 1999).
Alternatively, hypoxia-induced proteomic changes induce cancer dissemination
promoting cancer cells ability to adapt to nutrients deprivation or facilitating
proliferation, local invasion and/or metastatization in order to escape from a hostile
microenvironment.
As extensively debated in the next paragraph, cancer cells have developed elaborated
mechanisms in order to detect pO2 alteration and promote adaptation to hypoxia. The
master regulator of this complex system is represented by transcription factor HIF-1,
first identified by Semenza and collegues as a protein directly binding EPO promoter
DNA sequence (Wang et al. 1995). Its accumulation in response to low O2 level induces
activation of more than 100 genes involved in O2 delivery like erythropoietin,
angiogenesis (VEGF), anaerobic metabolism (glucose transporters and glycolitic
enzymes) and other key mechanisms involving survival and cancer diffusion (Vaupel et
al 2002; Höckel e Vaupel 2001).
60
HIF-1α (Hypoxia-Inducible Factor-1α)
HIF-1 is an hheterodimeric complex characterized by an α subunit of 120 kDa O2-
dependent (HIF-1α), and a β subunit of 91-94 kDa constitutively expressed (HIF-1β)
also known as aryl hydrocarbon nuclear translocator (ARNT), as it has been first
identified inside the hheterodimeric complex with the aryl hydrocarbon receptor (AHR)
(Reyes et al., 1992). Both subunits belong to the protein superfamily of basic helix-
loop-helix-Per/ARNT/Sim domains (bHLH-PAS) proteins (Wang et al., 1995)
containing a N-terminal bHLH domain and two PAS domains (PAS-A e PAS-B) in
order to specifically bind to DNA sequences called hypoxia responsive elements
(HREs) located in target genes promoters and to form the hheterodimeric complex HIF-
1α-HIF-1β, respectively (Crews 1998) (Fig. 15). At the C-terminus HIF-1α shows two
transactivation domains named N-terminal transactivation domain (N-TAD), between
aminoacids 531-575, and C-terminal transactivation domain (C-TAD), between
aminoacids 786-826 (Ruas et al., 2002). In particular, the latter interacts with specific
co-activators i order to activate genic transcription (Lando et al., 2002). In addition,
stability and degradation of HIF-1α subunit at different O2 tensions is regulated through
modifications of an O2–dependent degradation domain (ODDD) (Fig. 15).
Finally, nuclear localization sequences (NLS) have been identified both at the N-
terminal, between aminoacids 17-74, and at the C-terminal, between aminoacids 718-
721. These sequences are crucial for HIF-1α translocation into the nucleus independent
from dimerization with β subunit (Kallio et al. 1998).
Other proteins of the HIF family are HIF-2α, also known as endothelial PAS protein 1
(EPAS-1), HIF-like factor (HLF), HIF-related factor (HRF) and member of the PAS
superfamily 2 (MOP2) alternatively, (Ema et al., 1997; Flamme et al., 1997; Hogenesch
et al., 1997; Tian et al., 1997) and the HIF-3α factor (Fig. 17).
61
HIF-2α (Hypoxia-Inducible Factor-2α)
Subunit HIF-2, first named Endotelial PAS domain protein 1 (EPAS1) for its elevated
expression in endothelial cells and highly vascolarized tissues, has been first identified
through cDNA library screening in ovaric carcinoma HeLa cells. HIF-2 shows a 48%
sequence homology with HIF-1 and high conservation level of protein domains. As its
isoform 1, this protein belongs to the family of bHLH-PAS proteins. In the light of these
observations it was hypothesized that HIF-2 had similar, redundant biological roles to
HIF-1 (Kim et al. 2007). Both proteins share similar transactivation mechanisms O2–
dependent that involve both C-TAD and ODD domains. In particular, as in the case of
HIF-1, its O2–dependent regulation is related to ODD domain hydroxilation on Pro405
and Pro 531, which in turn drives HIF-1 to degradation via proteasome.
Fig. 17 HIF-1α, HIF-2α, HIF-3α1, IPAS e HIF-1β structures and functional domains.
62
Anyway HIF-1 and HIF-2 exert different biological roles according to their tissutal
expression and to N-TAD domain, which gives specificity for target genes. In fact while
HIF-1 is ubiquitously expressedd, HIF-2 expression is strictly related to lungs
endothelium and pneumocytes type II, cardiomyocites, duodenal epithelial cells and
interstitial cells in the kidneys (Gordan et al. 2007).
HIF-2 is complexed with subunit ARNT/HIF-2 in order to form the functionally
active hheterodimeric complex HIF-2/. In the nucleus HIF-2 binds to the same
DNA HRE sequence (5’-RCGTG-3’) as HIF-1.
Biological functions
As previously stated, HIF-1 and HIF-2 show different genic activation patterns. For
example, it has been observed that genes coding proteins related to cell metabolism are
almost all activated by HIF-1, while other genes like EPO are preferential targets for
HIF-2. In particular, HIF-2 activates renal and epathic production of EPO according
to hypoxic conditions in order to promote erithropoyesis.
In addition HIF-2α regulates iron homeostasis. An adjustment of iron metabolism is
needed to satisfy increased iron demand in the bone marrow. Cytochrome b (DcytB)
reduces ferric iron (Fe3+
) to its ferrous form (Fe2+
), which is then transported into the
cytosol of enterocytes by divalent metal transporter-1 (DMT1). DcytB and DMT1 are
both hypoxia inducible and HIF-2α regulated. Absorbed iron is released into the
circulation by ferroportin (FPN) and is then transported in complex with transferrin to
liver, reticuloendothelial cells, bone marrow, and other organs. Transferrin (Tf) is HIF
regulated, and hypoxia increases its serum levels. When intracellular iron levels are low,
iron regulatory protein (IRP) inhibits HIF-2α translation and diminishes hypoxia-
induced erythropoiesis (Haase 2010).
Several studies have also pointed out the role of HIF-2α in vascular tissues adaptation to
hypoxia for its higher transactivation ability on the promoter of the VEGF receptor
(VEGF-R) gene (Tian et al., 1997). Other genes regulated only by HIF-2α are for
example genes coding for embrional transcription factor Oct-4, cyclin D1, Twist1 and
TGF- (Patel e Simon 2008).
HIF-2 controls cellular proliferation is through modulation of c-Myc activity. C-Myc
promotes cellular proliferation by regulating the expression of genes involved in cell
cycle control including cyclins like cyclin D2 and cyclin kinase inhibitors like p21 and
p27. Unlike HIF-1α, HIF-2α promotes C-Myc-dependent activation of cyclin D2 and
63
repression of p27 in renal carcinoma cells. In particular, HIF-1α specifically disrupts c-
Myc/Max and c-Myc/Sp1 complexes, allowing more Mad/Max interaction and DNA
binding and causing cell cycle arrest in G1/S phase. On the other hand, HIF-2α
stabilizes c-Myc/Max complexes, in turn promoting c-Myc DNA binding at both E
boxes and Inrs. HIF-2 may also drive cell cycle progression through the activation of
cyclin D1. Cyclin D1 is a well-characterized cell cycle regulatory protein that is
upregulated in many cancers. Recent studies have shown a correlation between HIF-2-
mediated cyclin D1 expression and tumour growth in renal cancer cells (Loboda et al.
2010).
In addition HIF-2, but not HIF-1, is able to promote tumour growth in in vivo renal
cancer models. Stable overexpression of HIF-2 in renal cancer cells 786-O expressing
pVHL promote xenografts growth similar to 786-O deleted for pVHL, while stable
HIF-1 overexpression has the opposit effect on cancer cells growth. Additional studies
from the same research group also showed that only HIF-2 positively correlates with
the grade of displasia in pre-neoplastic lesions (Maranchie et al., 2002; Raval et al.,
2005).
The role of HIF-2 in promoting general tumourigenic mechanisms has also been
confirmed by Covello et al., who demonstrated that subcutaneous teratomas generated
from embrional stem cells (ESCs) ‘‘knocked in’’ for HIF-2 gene in the HIF-1 locus
show a fourfold higher xenograft growth in comparison to wild type cells (Covello et
al., 2005). This event is not only related to loss of HIF-1 expression as several studies
have pointed out that teratomas generated from ESCs HIF-1-/-
do not show the same
proliferative ability (Carmeliet et al., 1998). Finally, recent studies on human
neuroblastoma models have demonstrated HIF-2 stabilization in a condition of mild,
but prolonged, hypoxia, thereby suggesting that HIF-2 expression is able to promote
tumourigenesis even in tumours exposed to hypoxic stress of lesser extent (Holmquist-
Mengelbier et al., 2006).
64
HIF-3α (Hypoxia-Inducible Factor-3α)
HIF3α gene codificates for protein HIF-3, formed by 662 aminoacids with a 73 kDa
molecular weight. HIF-3α mRNA can be detected in a variety of tissues, including the
thymus, lung, brain, heart, kidney, liver, eye, and brain.
N-terminal bHLH-PAS domain shows homolgy sequence with HIF-1 e HIF-2
proteins of the 57% and the 53%, respectively, while HIF-3 C-terminal ODD domain
shows a sequence of 36 aminoacids with a 61% identity with HIF-1 ODD. In vitro
studies show that HIF-3 dimerizes with protein ARNT/HIF-3 similar to HIF-1 and
HIF-2 subunits. The resulting hheterodimeric complex HIF-3/ binds to HRE
sequence 5’-RCGTG-3’ thereby leading to activation of gene target expression.
In addition, there is a particular HIF-3 isoform which generates from alternative
splicing of the same mRNA as original HIF-3, first identified in murine models and
named inhibitory PAS domain protein (IPAS).
Biological functions
Biological functions of HIF-3 are still unclear, while there are evidences about IPAS
protein inhibitory role. IPAS do not show a C-TAD domain and does directly regulates
genic, but it semms to act as a direct negative dominant of HIF-1 and HIF-2, as it
binds to HIF-1α and HIF-2 N-terminal regions and prevent their binding to DNA
(Makino et al. 2001). In addition, alternative splicing IPAS-specific is hypoxia-
inducible in murine lung and cardiac tissues, thereby showing another O2-dipendent
post-translational modification of HIF- subunits. IPAS expression in normoxia has
been observed just in Purkinje neural cells and in epithelial corneal cells. In the latter,
IPAS seems to negatively regulate VEGF and angiogenic mechanisms (Makino et al.
2002).
65
Regulation of HIFs expression
Regulation of gene expression is similar for all proteins of the HIFs family. As for every
protein, HIFs expression is related to a balance between expression and degradation
mechanisms: in the case of HIF-1α, transcriptional and synthesis seem to be
constitutional mechanisms, while degradation is O2–dependent.
In normoxic conditions HIF-1α shows a very short half-life of about 1/2 ~ 5 minutes
and is rapidly degradated via ubiquitin-proteasome system (Salceda et al., 1997). When
cells are exposed to low O2 tensions, HIF-1α half-life is of about 30 minutes, the protein
is stabilized and traslocates to the nucleus, where it binds to subunit HIF-1β forming the
transcriptionally active HIF complex. The resulting heterodimer binds to HRE sequence of
target genes and binds to transcriptional coactivators, thereby promoting gene expression
(Lando et al., 2002).
HIF-1α stability and transactivation ability are principally regulated by post-
translational modifications on HIF-1α domains, like hydroxylation, ubiquitination,
acetylation and phosphorilation (Brahimi-Horn C et al., 2005).
This complex regulation mechanism allows cells to quickly adapt to variations of O2
concentration accrding to its availability. HIF-1α degradation in normoxic conditions is
mainly mediated by ODD domain hydroxilation and the reaction is catalyzed by
specific enzymes called prolyl hydroxylases-domain proteins (PHDs) (Huang et al.,
1998).
The prolyl hydroxylase-domain proteins (PHDs)
In normoxic conditions, newly sintetized HIF-1α is quickly hydroxylated on Pro 402
and 564 located in the ODD domain. This reaction is catalyzed by three enzymes known
as prolyl hydroxylase-domain protein (PHDs) 1-3. Pro 402 and 564 are highly
conserved in HIF-2α (Pro405 e Pro530) and HIF-3α proteins and they are located inside
a highly conserved consensus sequence, LXXLAP. Only HIF-3α shows a different
aminoacidic sequence, LXXLHP (Bruick et al., 2001; Masson et al., 2001).
PHDs are dioxygenases 2-oxoglutarate-dependent which use O2 and α-ketoglutarate as
substrates and Fe2+
and ascorbate as cofactors. The hydroxilation mechanism is
characterized by scission of molecular O2 and one atom is transferred on a Pro residue,
while the other one reacts with α-ketoglutarate, thereby creating succinate and CO2
(Bruick RK et al., 2001). In physiologic conditions O2 is a limiting substrate and this
mediates HIF-1α O2-dependent regulation (Jiang et al., 1996).
66
All three PHD isoforms hydroxylate HIF-α in vitro, but PHD2 shows higher activity
(attività relativa PHD2>>PHD3>PHD1) and it has been demonstrated as the master
regulator of HIF-1α turnover in vivo (Huang et al. 2002). According to these data,
PHD2 gene silencing induces HIF-1α stabilization in normoxia, while PHD1 e PHD3
inhibition does not show same effect (Berra et al. 2003). In addition, hypoxia induces
both PHD2 and PHD3 expression, but it does not seem to have effects on PHD1
(Epstein et al. 2001). This could represent a mechanism of regulation of HIF-1α activity
induced by HIF-1α itself. According to this hypothesis, PHD2 is mainly located in the
cytoplasm, while PHD1 is mainly expressed in the nucleus and PHD3 in both
compartments. Anyway, PHD2 is able to translocate to the nucleus, thereby
contributing to HIF-1α degradation in both nucleus and cytoplasm (Metzen et al. 2003).
Pro-402 and Pro-564 hydroxylation is a key event for HIF-1α degradation because it
allows the interaction between oncosuppressor von-Hippen-Lindau (pVHL) protein and
hydroxylated HIF-1α ODD domain (Ivan et al. 2001). pVHL is a component of the E3
ubiquitin-ligase complex which drives protein digestion via proteasome (Cockman et
al., 2000). In fact pharmacological inhibitors of proteasome activity and/or mutations of
E1 enzyme stabylize HIF-1α, showing the importance of ubiquitination and proteasome
in normoxic HIF-1α degradation (Huang et al. 1998; Salceda et al. 1997). After
hydroxylation and binding to pVHL protein, the complex associates to proteins elongin
C, elongin B cullin-2 and Rbx-1, forming the complex VCB-Cul2 E3 ligase. HIF-1α
interaction with this multiproteic complex induces HIF-1α poliubiquitination, leading to
HIF-1α proteosomic digestion (Kamura et al. 2000).
pVHL is an oncosuppressor protein whose mutations are involved in development of
several types of cancer (Iliopoulos et al., 1998; Schoenfeld et al., 1998). This protein
was first characterized in the von Hippen-Lindau syndrome, hereditary disease with
development of multiple tumours in several body tissues. Cells with mutated pVHL
show more stable and active HIF-1α e HIF-2α in normoxic conditions, thereby causing
an overexpression of hypoxia-induced genes and promoting cancer progression
(Iliopoulos et al. 1996). The complex pVHL- E3 ligase is ubiquitary expressedd in
different tissues and it is preferentially located in the cytoplasm, but like PHD2 is able
to translocate to the nucleus, promoting HIF-1α degradation in both compartments in
normoxia (Berra et al. 2001). On the contrary, low O2 tension directly inhibits PHDs
activity because the consequent alteration of the mitochondrial chain transport leads to
an excess of reactive oxygen species (ROS). ROS in turn change the oxidation state of
67
iron inside PHDs active site from Fe2+
to Fe3+
. Subsequently ferric iron is no longer
active (Epstein et al. 2001; Simon 2006). In this condition, HIF-1α is no longer
hydroxylated and led to degradation, but stabilized and accumulated (Fig. 18). In
addition, PHD activity can be regulated by intracellular Ca2+
concentration (Berchner-
Pfannschmidt et al. 2004) and E3 ubiquitin-ligase Siah1 e Siah2 enzymes, whose
trascription is hypoxia-induced (Nakayama e Ronai 2004).
Inside the HIF-1α ODD domain, Lys 532 can be acetylated by an acetyl-transferase
enzyme named arrest-defective 1 (ARD1) (Jeong et al. 2002). Lys532 acetylation
promotes HIF-1α-pVHL interaction, thereby destabilizing HIF-1α. According to the
importance of this post-translatinal modification, Lys 532 mutation in Arg 532 induces
HIF-1α stabilization (Tanimoto et al. 2000). In addition, when cells accumulate butirric
acid, general inhibitor of deacetylases, there is an increase in HIF-1α acetylated state
leading to significative reduction of expression (Kim et al., 2001). As acetyl transferases
is not influenced by O2 tension, ARD1 can be active and acetylate HIF-1α in an O2-
independent manner, even if same studies have shown that ARD1 mRNA is less
expressed in hypoxia, thereby leading to a reduction of HIF-1α acetylation in
comparison with normoxic condition (Jeong et al. 2002).
Factor inhibiting HIF (FIH)
Despite post-translational modifications, HIF-1α stabilization alone is not enough to
guarantee its activity as transcriptional activator.
Another key mechanism for HIF-1α activity regulation is modulation of transactivation
domains N-TAD and C-TAD. These two domains are separated from each other by an
inhibition domain (ID) located between aminoacids 576-785, which downregulates their
function in presence of high O2 tension (Jiang et al., 1997-b).
Regulation of transcriptional activity is mediated by a molecular mechanism different,
but correlated to the one that influences HIF-α stability in hypoxia. In normoxic
condition, hydroxylation of Asn803 in HIF-1α and Asn851 in HIF-2α induces a steric
inhibition of interaction between HIF-1α and its coactivator CBP/p300, impairing its
recruitment that is necessary for HIF-1α activity (Sang et al., 2002). In hypoxic
condition there is not Asn hydroxylation, so HIF-1α can associate to CBP/p300
throucgh C-TAD domain in order to activate target genes transcription (Lando D et al.,
2002).
68
This molecular regulation is mediated by an asparagyl-hydroxylase enzyme named
factor inhibiting HIF-1 (FIH-1) (Mahon et al., 2001). As PHDs, also FIH-1 is a
dyoxygenase 2-oxogluarate-dependent and has Fe2+
and ascorbate as cofactors and O2
as a substrate, making this enzyme a proper oxygen levels sensor. FIH-1 is primarily
located in the cytoplasm, but there is also a little nuclear fraction whose transcription is
O2-dependent and that regulates HIF-1α activity rather than its stability (Metzen et al.,
2003). According to this, structural analysis have demonstrated that, when HIF-1α is
complexed to CBP/p300, Asn 803 is buried inside the proteic complex in order to
prevent its hydroxylation. In fact, mutation of Asn 803 with Ala 803 allows interaction
between HIF-1α and its coactivators even in presence of high O2 tension (Dames et al.,
2002; Freedman et al., 2002; Lando et al., 2002). Although interaction of FIH-1 with
pVHL is not required for its activity, it has been demonstrated that pVHL binds to FIH-
1, forming a complex with HIF-1α (Mahon et al., 2001). This allows hystone
deacytilases recruited by pVHL to interfere with the transcriptional process, thereby
promoting FIH-1 inhibitory acivity on HIF-1α (Hewitson et al., 2002; Sang et al., 2002)
(Fig. 18).
Fig. 18 Regulation of HIFs expression in normoxia and hypoxia.
69
In the same way as PHD, FIH-1 activity depends not only upon O2 and 2-oxoglutarate
availability, but also requires cofactors Fe2+
and ascorbate, while its transcription is O2-
independent (Lando et al., 2002; Metzen et al., 2003). FIH-1 structural analysis have
pointed out that FIH-1 requires formation o fan omodimeric complex in order to bind its
substrates and to perform its catalytical activity. As O2 availability is a limiting factor
for FIH-1 activity, it has been hypothesized that this enzyme has a role in sensing
extracellular O2.
In conclusion, O2-dependent HIF-1α functional regulational depends on “two steps”
model: i) O2-dependent prolyl-hydroxylation in the ODDD domain in order to prevent
association with the multiproteic complex E3 ubiquitin ligase and subsequent
proteasomal degradation; ii) O2-dependent inhibition of Asn hydoxylation in the C-
TAD domain to allow interaction with coactivator with CBP/p300 and functionally
active complex formation (Fig. 18).
Sirtuins (SIRTs)
Sirtuins are a family of hystone-deacetylases enzymes NAD+-dependent wich have
several roles in regulating genic transcription, DNA repair and cell metabolism. These
enzymes respond to variations of the NAD+ oxidized/ NADH reduced ratioand
represent important redox intracellular sensors (Denu, 2003). In mammalians there are
seven homologous of yeast Sir2 protein: SirT1, 6 e 7 proteins are located in the nucleus,
SirT2 is expressed in the cytoplasm, while SirT3, 4 e 5 are strictly located in the
mitochondria (Haigis e Guarente 2006).
SirT1 has been recently described as a negative regulator of HIF-1α activity through its
direct acetylation. This regulation is independent from intracellular levels of NAD+ e
NADH, as when hypoxia increases NADH level, HIF-1α deacetylation SirT1-mediated
is downregulated (Lim et al. 2010).
SirT1 also regulates HIF-2α transcriptional activity through deacetylation. In particular,
it has been demonstrated that SirT1 directly binds to HIF2α, but not to HIF1α, and
deacetylates specific lysin residues. For example, Sirt1 co-localizes with HIF2α at the
Epo gene promoter, thereby stimulationg HIF2α pro-transcriptional activity and
promoting epathic and renal EPO production both in in vitro and in vivo models. These
data demonstrate that EPO production is dependent from both O2 availabilty and
cellular redox state (Dioum et al. 2009) (Fig. 19).
70
Fig. 19 HIF-2α stabilization by SirT1 activity.
Recently it has been demonstrated that also SirT6 can inhibit HIF-1α-mediated genic
transcription through direct binding to HREs sequences and subsequent hystone
deacetylation in promoters of genes related to cell metabolism. According to that cells
Sirt6−/− show incresed expression of glycolitic enzymes and increased glucose
consumption, both in vitro and in vivo models (Zhong et al. 2010).
In the last years also SirT3 has been subject of numerous studies. In particular, Kim and
collegues have demonstrated that this protein acts as a mitochondrial oncosuppressor
gene. In a model of embrional murine fibroblasts ingegnerized to express oncogenes
RAS and Myc, total deletion of SirT3 is sufficient to promote neoplastic transformation
and tumour development in murine models. In addition, transformed cells show a
glycolitic metabolic behaviour, with increased glycolisis and oxidative stress and
oxidative phosphorilation inhibition. According to that, excess ROS production due to
loss of SirT3 expression promotes DNA damage process and thus accelerates cancer
progression (Kim et al. 2010).
The role of SirT3 as oncosuppressor gene has been confirmed by Bell and collegues
colleghi who have pointed out that loss of SirT3 promotes murine embrional fibroblasts
proliferation in hypoxic condition. Cells Sirt3−/−
show higher ROS levels compared to
control cells, thereby favouring HIF-1α stabilization throught inhibition of PHDs
71
activity. These data have been then confirmed reverting HIF-1α stabilization following
both antioxidants administration like N-acetyl-cysteyn (NAC) and SirT3 (Bell et al.
2011) (fig. 20).
Fig. 20 Regulation of HIF-1α stabilization by SirT3.
Phosphorilation, nitrosation and sumoilation of HIF-α
An other molecular mechanism that regulates HIF-1 stability in a PHDs independent
manne ris represented by molecular chaperon Hsp90, which acts independently from
pVHL expression and O2 tension (Isaacs et al., 2002).
In addition, it has been described a HIF-1α regulation through direct phosphorilation by
mytogen-activated protein kinases (MAPK), where both Erk1/2 and p38 phosphorilates
both HIF-1α and HIF-2α, as observed in in vitro and in vivo models (Richard et al.,
1999). This post-translational modification does not affect HIF-1α stability or its DNA
binding ability, but positively regulates its transcriptional activity. The most accepted
explanation for this event is that HIF-1β subunit preferentially binds to phosphorilated
HIF-1α subunit, thereby leading to increased HIF-1α activation and target genes
transcription like VEGF and EPO (Richard et al., 1999).
Other described post-translational modifications regulating HIF-1α are S-nitrosation on
Cys800, which promotes direct interaction with coactivator CBP/p300, thus promoting
HIF-1α transcriptional ability, and SUMOilation, which affects both degradation and
protein stability of HIF-1α (Yasinska et al., 2003).
72
In particular, hypoxia promotes expression of the small ubiquitin-like modifier-1
(SUMO) protein, thereby favouring HIF-1α sumoilation (Shao et al., 2004). At the
beginning it was hypothesized that this modification could improve protein stability,
while recent observations have demonstrated that this molecular mechanism promotes
HIF-1α degradation by promoting HIF-1α binding to pVHL-E3 ligase complex. This
regulatory mechanism is completely independent from PHDs hydroxylation state.
In hypoxic condition, HIF-1α degradation SUMO-mediated is inhibited by expression
of SUMO1/sentrin specific peptidase 1 (SENP1), a nuclear protease that removes the
SUMO protein from HIF-1α. As a demonstration of the importance of this mechanism
for HIF-1α stabilization, cell lines knock-out for SENP1 gene show reduced HIF-1α
expression. According to these observations, SENP1-/-
murine fetus showed a severe
fetale anemia caused by failing regulation of EPO expression, thereby showing SENP1
physiological role in regulating HIF-1α (Cheng et al., 2007).
Regulation of HIF activity and expression by growth factors
HIF-1α expression induced by growth factors stimulation differ from HIF-1α hypoxia-
induced expression because it is not associated to a decreased degradation, but to an
increased production through activation of phosphatydil-inositol-3-kinase (PI3K) and
MAPK pathways (Fukuda et al., 2002; Fukuda et al., 2003; Laughner et al., 2001;
Zhong et al., 2000). According to that, cell stimulation with molecules like the
epidermal growth factor (EGF), fibroblast growth factor 2 (FGF-2), insulin and insulin-
like growth factors 1 and 2 (IGF-1-2) and IL-1β promote HIF-1α expression, DNA
binding and target genes activation even in normoxic condition (Feldser et al 1999;
Hellwig-Burgell et al 1999; Laughner et al 2001; Zelzer et al 1998). Binding of these
factors to their tyrosine-kinase receptors activates several molecular pathways like te
MAPK and PI3K ones.
MAPK pathway leads to activation of both ERK1-2, also named p42/p44 MAPK, and
p38 MAPK, following previous activation of Ras/Raf-1/MEK-1/ERK1-2. It has been
demonstrated that HIF-1α is phosphorilated by p42, p44, p38α and p38γ in vitro, but
aminoacids involved in this modifications have not been discovered yet (Richard et al
1999; Sodhi et al 2000).
PI3K pathway directly activates Akt kinase (protein kinase B), which is related to
several cellular mechanisms like apoptosis, cell cycle regulation and proteic translation
(Vivanco e Sawyers 2002). One of Akt target proteins is FRAP, protein associated to
73
rapamicyn FKBP12 (mTOR) which activates ribosomal protein p70S6
kinase (p70s6k
)
and promotes mRNA translation with polipyrimidinic sequences at 5' end (Brown et al
1995; Zhong et al 2000). In addition, Akt also phosphorilates 4E-binding protein (4E-
BP), known transcriptional regulator, its hyperphosphorilation leading to an increased
proteic translation. This molecular pathway is negatively regulated by tumour
suppressor phospatase end tensin homologue (PTEN) coding for a phosphatase that,
dephosphorilating PI3K substrates, downregulates all PI3K-AKT-FRAP pathway (Fig.
21). Differing from hypoxia which upregulates HIF-1α expression in all cell types,
growth factors stimulation induces HIF-1α expression just in certain cell lines.
Fig. 21 Molecular pathway of HIF-1α
expression growth factors-induced.
74
It is now well understood how inflammation represents one of the key events of cancer
development. Recent studies have thus suggested the role of HIF-1α in regulating
inflammatory mechanisms, showing that IL-1β positively regulates normoxic HIF-1α
expression and promotes the HIF-1α-dependent VEGF production through activation of
the nuclear factor-κB (NF-κB) molecular pathway (Jung et al. 2003). Anyway, IL-1β
also mediates HIF-1α expression through a post-translational mechanism that blocks the
pVHL-dependent HIF-1α degradation, thereby leading to HIF-1α stabilization (Jung et
al. 2003). According to that, HIF-1α is activated in normoxia through genetic alterations
of O2-related signaling pathways and pVHL has a central role in regulating HIF-1α
transcriptional activity (Rankin and Giaccia 2008).
Cicloxygenase-2 (COX-2)-dependent NF-κB expression mediated by IL-1β is one of
the HIF-1α positive effectors genes. In fact, it has been demonstrated that IL-1β
positively regulates HIF-1α through activation of the classical inflammatory pathway
NF-κB and COX-2-mediated that leads to VEGF activation, tumour and metastasis
growth (Yung et al. 2003). Despite reduction of IL-1-mediated HIF-1α induction after
administration of COX-2 inhibitors, prostaglandin E2 (PGE2), pysiological product of
COX-2 activity, promotes HIF-1α expression in a dose-dependent manner.
In conclusion, in hypoxic condition, PHDs and FIH are inactive and HIF-1α can
positively modulate expression of its target genes.
Reactive oxygen species (ROS)
Reactive oxygen species (ROS) are O2 partially reduced metabolites, showing higher
reactivity than O2 itself. Major ROS produced within the cell are superoxide anion
(O2־·), hydroxyl radical (OH·), nitrogen monoxide (NO) and oxygen peroxide (H2O2).
Thanks to their higher reactivity, excessive amounts of these molecules can severly
impair cell functions. Physiologically, high ROS production is first related to cells
involved in immunitary defense, as transient ROS production promotes expression of
chemokines, cytokines and endothelial/leukocytic adhesion molecules, thereby
activating anti-inflammatory response like elimination of bacterial/viral exogen
pathogenic agents (Remick and Villarete 1996). As production of ROS can be highly
toxic for cells and neighboring tissues, their production must be severly regulated
through various molecular mechanisms.
Several studies have then showed that ROS can have an important role also in cells not
related to immunitary system. In particular, it has been hypothesized that ROS can act
75
as second messengers in almost all cellular pathways like genic expression,
celldifferentiation, apoptosis, proliferation and cell adhesion (Poli et al 2004; Aslan and
Ozben 2003; Chiarugi 2005; Chiarugi 2001; Giannoni et al 2005). On the other hand,
aberrant signaling ROS-dependent leads to pathologic events like cell cycle
disregulation (Boonstra and Post 2004), apoptosis and senescence (Gourlay and
Ayscough 2005; Johan et al 2005), ischemic/reperfusion events (Otani 2004) and
diabetis-related complications (Niedowiez and Daleke 2005).
Cellular energetic metabolism is based upon ATP production through the activity of the
mitochondrial electron tran sport wich leads to complete O2 reduction into H2O by
accepting electrons and H+
ions. During oxidative phosphorilation in the mitochondrial
inner membrane, electrons are transferred from molecular carriers like nicotinammide
adenin dinucleotide (NADH) or flavinadenin dinucleotide FADH2 to the electron
transport chain, compsed by three enzymatic transmembrane complexes which
contribute to produce a transmembrane proton electrochemical gradient. If protons flow
back through the membrane, they enable mechanical work, such as rotating
bacterial flagella. ATP synthase, an enzyme highly conserved among all domains of
life, converts this mechanical work into chemical energy by producing ATP from ADP
and Pi (inorganic phosphate) (fig. 22).
During these complex series of redox reactions, a single electron can be directly
transferred to O2, thereby producing O2־· in the complex I (NADH/ubiquinone
oxidorecuctase) and complex III (ubiquinol/cytocrome c oxidoreductase) (fig. 16). The
electrons chain transport is a highly efficient mechanism and usually consume the
majority of O2 available. Anyway, it has been clearly demonstrated that almost 1-2% of
all transported electrons are lost by transmembrane complexes, thereby generating O2־·
through reactions catalyzed by Q coenzyme and ubiquinone and related complexes
(Forman and Boveris 1982). According to their role in aerobic cellular respiration,
mitochondria are major ROS producers in vivo (Boveris e Chance 1973).
Another electron chain transport is located on the endoplasmic reticulum, where O2־· is
produced from nicotin amide adenin dinucleotide-3-phosphate (NADPH) cytocrome
P450 oxidoreductase (Cross e James 1991). Anyway, O2־· can be generated also from
ipoxantine/xantine oxidase, NADPH oxidase, lipoxigenase (5-LOX) and COX enzymes.
Also radiant energy absorption can generate ROS as the ionizing radiations like UV and
γ rays are able to hydrolyze H2O into OH· and radicalic hydrogen (H·) (fig. 22).
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HIF-α proteins can be stabilized in a ROS-dependent manner. Several evidences have
showed that inhibition of mitochondrial ROS productions strongly impairs HIF-α
stabilization in hypoxic condition (Chandel et al 1998; Weinberg and Chandel 2009). In
agreement with these observations, molecules used in therapy can promote O2
redistribuction in the cytoplasm, outside the miotchondria, thereby leading to increased
PHDs activity even in conditions of mild hypoxia (Hagen et al 2003; Vaux et al 2001).
In addition, genetic studies demonstrate that complex III disruption, cytocrome C and
Rieske Fe-Cu protein block HIF hypoxic stabilization (Mansfield et al 2005; Guzy et al
2005), while disruption of complex IV do not has the same result (Brunelle et al 2005).
These results suggest that cellular respiration is not required for HIF stabilization, while
ROS generated from the electron transport chain play a fundamental role in this event.
ROS-dependent HIF-1α stabilization is mainly related to modulation of PHDs activity,
as they need oxygen, oxoglutarate and Fe2+
in order to fully sustain their activity. In
fact, it has been demonstrated that PHDs regulation Fe-dependent is mediated by ROS
in a model of junD -/-
cells, characterized by a condition of chronic oxidative stress
(Gerald et al., 2004). According to this model, ROS production interferes with Fe2+
availability in the prolyl-hydroxilasic cathalitic site, thereby impairing PHDs activity in
normoxic conditions. In fact, high H2O2 levels promote Fe2+
oxidation to Fe3+
, with a
consequent increase of Fe3+
, inactive intracellular PHDs percentage. Subsequently,
Fig. 22 Molecular pathways of ROS production.
77
exogenous addicition of Fe2+
in junD-/-
cells strongly reduces HIF-1α levels. The same
effect is obtained after admnistration of reducing agents like cysteine or gluthatione, or
antioxidants molecules like ascorbate, whose physiological concentrations increase
PHDs activity and thus HIF-1α degradation, reverting the oxidation state of the atom of
Fe in PHDs catalytic site (Knowles et al., 2003).
Like PHDs, also FIH activity is highly O2, oxoglutarate and Fe2+
-dependent, thus also
FIH is highly sensitive to ROS-induced Fe3+
oxidation state, thereby promoting HIF-1α
accumulation. In addition it has been showed that some glucose metabolites like the 2-
oxiacids can directly interact with PHDs oxoglutarate-binding domain, impairing their
enzymatic activity. In addition, these molecules also change the catalytic Fe oxidation
state, as this inhibitory effect 2-oxiacids-mediated is fully reversed after exogenous
administration of Fe2+
,ascorbate and/or reducing agents (Lu et al., 2005) (Fig. 23).
Fig. 23 HIF-1α stabilization thorugh ROS-mediated PHDs inactivation.
Anyway, molecular mechanism by which hypoxia causes an increase in ROS
production has not been clearly understood yet. From a thermodynamic point of view it
78
seems unlikely redudec substrate concentration would cause an increase in the rate of
the electron transport chain, thus it has been hypothesized that other factors may interact
with these mechanism.
According to the ‘vectoral transport’ hypothesis, ROS lacking from complex III can be
released to either the matrix side of the membrane or towards the intermembrane space
(Muller et al. 2004). Thus O2 dissolved in the lipid bilayer might affect the ROS
balance, thereby leading to higher oxidants release to the intermembrane space direction
than to the matrix side. Although the specific mechanism responsible for this shift has
not been elucidated yet, an oxygen-dependent change in the direction of ROS release
could explain oxidant stress increases in the cytosol during hypoxic conditions.
Interestingly, such a shift could theoretically rely on an increase in cytosolic ROS
signalling even if overall ROS production were decreased.
The ‘semiquinone lifetime’ hypothesis proposes that O2 interaction with protein or
lipids at complex III could regulate the lifetime of ubisemiquinone at the Qo or Qi sites.
Reduced electron removal from ubisemiquinone by the b cytochromes during hypoxia
could subsequently promote superoxide production even under lower O2 concentrations.
Any small molecule or drug that alters the kinetics of electron removal from the
semiquinone to the b cytochromes can potentially affect superoxide generation (Guzy e
Schumacker 2006).
Finally, the ‘oxygen access’ hypothesis suggests that hypoxia might increase the access
of O2 to the semiquinone moiety at complex III. According to this hypothesis, if the
molecular structure of one or more proteins in complex III were affected by the level of
oxygen in the membrane, the ability of O2 to attack the semiquinone were improved
under low oxygen conditions and this could lead to an increase in ROS production, even
under decreased O2 availability.
Anyway, all these models require a modification of lipid–protein structure mediated by
molecular oxygen, or a change in the concentration of oxygen, thereby increasing the
transfer of an electron from ubisemiquinone to O2 despite the lowered concentration of
oxygen itself (Guzy e Schumacker 2006).
79
HIFs target genes
As stated in previous paragraphs, HIF-1α is the master regulator of processes related to
cellular hypoxic response like erythropoiesis, angiogenesis and glycolitic metabolism.
In hypoxic conditions, complex HIF-1α/β interacts in the nucleus with its coactivators
like p300 or CBP (Ruas et al., 2005) and regulates genes transcription after binding to
HREs DNA sequences (Kasper et al., 2005; Kasper et al., 2006).
Given the role of HIF-1α in mediating also cancer-related pathological processes, the
study of genes and molecular pathways activated by HIF is becoming more and more
important in order to find always new therapeutic anti-cancer therapies (Fig. 24).
Fig. 24 HIF-1 target genes: a complex network.
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Angiogenesis
Angiogenesis is a key process for tumour growth and dissemination and is characterized
by recruitment and proliferation of endothelial cells from the stroma to the tumour
microenvironement. The ability of cancer cells to metastatize distant tissues is
positively correlated to tumour angiogenic potential, thereby leading to formation of
new vessels in order to promote cancer cells dissemination in the bloodstream (Hill
1990; Weidner 1993; Weidner et al 1991). At the same time tumours also exploit
lymphoangiogenesis to favour tumour growth also through lymphatic vessels. In
addition, new vessels can originate from endothelial and tumour cells or from tumoural,
non-endothelial cells through a process known as “vascular mimicry”.
VEGF is the most powerful and selective endothelial mitogenic agent. It induces a
quick and complete angiogenic response through interactions with its receptors,
VEGFR-1/Flt-1 e VGFR-2/Flk-1/KDR, expressedd on pre-existent endothelial cells,
thereby leading to cells proliferation and migration (Leung et al 1989). High VEGF
levels are present in several types of solid tumours and are positively correlated to
increased cancer vascularization, metastatic ability, resi stance to terapie and bad
prognosis (Toi et al 1996; Takahashi et al 1995; Toi et al 1994).
As already stated, hypoxia induces VEGF production through several molecular
mechanisms like increased protein and mRNA stability. According to that, HREs have
been identified in both 3' and 5' regions flanking the VEGF human gene (Michenko et al
1994; Forsythe et al 1996; Levy et al 1996; Mizukami et al 2004), and its expression
induced by hypoxia involves both HIF-dependent (Forsythe et al 1996; Shinojima et al
2007) and HIF-independent (Mizukami et al. 2004) molecular pathways. Anyway,
VEGF expression and tumour vessels density in vivo are primarly correlated to a HIF-
dependent mechanism wich can be highly influenced by several factors like genic
mutations and the peculiar characteristics of tumour microenvironment (Carmeliet al
1998).
Several studies have pointed out how several genes correlated to different phases of
angiogenesis are more expressedd under low O2 tension and thus are controlled by HIF-
1α expression (Levy et al., 1995; Bunn e Poyton, 1996; Forsythe et al., 1996; Berra et
al., 2000; Giordano e Johnson, 2001; Semenza, 2002). In fact HIF-1α can regulate
expression of both pro-angiogenic cytokines/growth factors and of their respective
receptors. Among them the most important are stromal-derived factor-1 (SDF-1) and its
receptor CXCR4 (Staller et al., 2003), placentar growth factor (PLGF), angiopoietin 1
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and 2, platelet-drived growth factor-B (PDGF-B), VEGF and its receptor VEGF-R1 also
known as Flt-1 (Fms-like tyrosine kinase-1), which binds both PLGF and VEGF
(Gerber et al., 1997; Okuyama et al., 2006). Those ligands directly interact with their
receptor expressed on the membrane of endothelial cells and endothelial progenitors,
smooth muscle cells, mesenchimal stem cells and other cells bone marrow-derived
(Bosch-Marce et al., 2007; Ceradini et al., 2004; Forsythe et al., 1996; Kelly et al.,
2003; Simon MP et al., 2008), thereby promoting their recruitment and
proliferation/vessels organization in the avascularized tumour areas (Neufeld et al.,
1999; Jośko et al., 2000; Conway et al., 2001).
Expression of VEGF and other pro-angiogenic factors induces increased vascular
density, facilitating O2 and nutrients diffusion in all cancer areas. In addition, HIF-1α
activates genes related to control of vessels muscolar tone like nitric oxyde syntase-2
(NOS-2) (Melillo et al., 1995), heme-oxygenase 1 (Lee et al., 1997), endothelin 1 (ET1)
(Hu et al., 1998), adrenomedulin (ADM) (Nguyen e Claycomb, 1999) and α1B
adrenergic receptor (Eckhart et al., 1997). Finally, in hypoxic condition, there is also a
general upregulation of all proteins involved in ECM degradation and vascular
maturation like MMPs (Ben-Yosef et al., 2002), plasminogen activator, its receptors and
its molecular inhibitors (PAIs) (Kietzmann et al., 1999).
Invasion and cell motility
After acquiring a metastatic phenotype, neoplastic cells are still physically separated
from neighbouring tissues by basal membrane and ECM. In order to avoid this barrier,
cells need to digest matrix proteins through the expression of protelytic enzymes like
the MMPs family proteins. These enzymes degrade all the main components of
interstitial matrix and basal membrane, thereby allowing cancer cells penetration and
diffusion in distant tissues (Brinckerhoff e Matrisian 2002).
MMPs are zinc-dependent endopeptidases ubiquitously expressed in human tissues and
associated to cancer metastatic ability. They are divided into five groups according to
their substrate: matrilysines, collagenases, stromalysins, gelatinases and membranes
MMPs. These enzymes are also classified according to their structures in eight groups,
five of them are secreted and three are bound to the cell membrane.
MMPs are sintethyzed as pre-pro-peptides containing both zinc and calcium ions. Their
structures contain an amino-terminal pre-peptide, or signal peptide, of 17-29 aminoacids
which directs the protein to the endoplasmic reticulum; a catalytic domain of 170
82
aminoacids with a zinc binding domain; a pro-peptide of 77-87 aminoacids containing a
thiolic group (SH) which interacts with zinc and keeps the enzyme into an inactive
form. In the majority of the MMPs, the cysteine residue is in the conserved
sequence PRCGxPD. Some MMPs have a prohormone convertase cleavage site Furin-
like as part of this domain, which, when cleaved, activates the enzyme.
Recent studies have demonstrated that the role of MMPs in cancer development is way
more complex than the simple degradation of matrix proteins (Hojilla et al 2003).
According to that MMPs act on several types of substrates including growth factors
receptors, adhesion molecules, chemokines, cytokines, apoptotic ligands and angiogenic
factors, thereby influencing most of the cancer progression phases.
MMP-2 and MMP-9, also named gelatinase A and B respectively, are the major MMPs
implicated in tumour progression as key enzymes for collagen IV digestion, wich is the
main compnent of ECM (Stamenkovic 2003). Sperimental evidences suggest that there
is a strong correlation between MMPs increased expression and tumour cells invasive
ability (Vihinen e Kahari 2002) and studies conducted on a pulmonar epithelial cells
revealed that they are upregulated in hypoxia in a HIF-dependent manner (Leufgen et al
2005).
ECM digestion also requires activity of uPA/uPAR complex where uPA is activated
after interaction with its receptor uPAR. Active uPA turns plasminogen in plasmin
through proteolytic cut. Plasmin is directly involved in ECM proteins degradation and
activation of several growth factors and MMPs which in turn contribute to further
degrade ECM. In addicition, uPAR can regulate other memebrane proteins like
integrins, growth factor receptors and receptors coupled with G protins in order to
promote cell migration (Yebra et al 1996). For example, uPAR acts as receptor for
vitronectin to promote cell adhesion to ECM (Waltz e Chapman 1994), while
interaction between uPAR and β1 and β2 integrins impairs cell adhesion (Wei et al
1996). In hypoxic condition increased uPAR mRNA expression results from an
increased mRNA stability and higher activity of its gene promoter (Maity et al 2000). In
addition, experimental evidences have pointed out a positive correlation between HIF
and uPAR expression as HREs are located in the upper domain of uPAR promoter
sequence (Graham et al 1998) and both uPAR expression and in vitro invasive ability of
colon carcinoma cells are increased by HIF-1α overexpression and thus impaired by its
gene silencing (Krishnamachary et al 2003).
83
Anyway, there are also several pathways HIF-independent involving inhibition of NO
production, activation of soluble guanilate cyclase and protein kinase G cGMP-
dependent (PKG) (Postovit et al. 2002) or activation of MAPK like ERK1/2 and p38
(Yoon et al 2006). In order to escape from a hostile hypoxic microenvironment, cancer
cells exploit migrating strategies in order to move toward higher availabilty of oxygen
and nutrients. For exemple, HIF-1α activation correlates with metastatization through
direct regulation of key factors governing tumor cell metastatic potential, including
Snail-dependent downregulation of E-cadherin expression and loss of cell-to-cell
contact, lysyl oxidase (LOX) activation, and upregulation of both stromal-derived factor
1 (SDF-1) and its receptor CXCR4, thereby directly regulating metastatic cancer cells
directional migration (Rankin and Giaccia, 2008). In addition, some factors like HGF
play a mandatory role in mediating HIF-1α-dependent cancer cells invasiveness.
HGF is a cytokine secreted from stromal cells which binds to its high specific receptor,
coded by the oncogene MET, which expression is induced in hypoxia in several types of
cencers. Several studies have demonstrated that hypoxia has a major role in promoting
MET expression, leading to higher metastatization in vivo and higher invasivity in vitro.
Hypoxic upregulation of MET promoter, its expression and phosphorilation have been
observed in both cell lines derived from hypoxic and normal tissues, even if its
expression and HGF production is increased by interaction between cancer cells and
hypoxic tissues-derived fibroblasts (Ide et al 2006). In addition, it has been
demonstrated that hypoxic MET expression is related to HIF-1 binding to two sites
located at the 5' terminus of the non coding region of MET promoter Met (Pennacchietti
et al 2006). According to that, the synergic effect between hypoxia and HGF production
on cancer cells invasive ability is impaired after HIF-1 gene silencing (Hara et al 2006).
A positive feedback regulatory mechanism contributes in sustaining and amplifing MET
overexpression in solid tumours, as MET molecular pathways induce both HIF-1α and
MET itself activity (Boccaccio et al 1994; Tacchini et al 2001; Tacchini et al 2003).
Met is a tyrosine kinase receptor first expressedd as a single chain precursor, which is
then cleaved at a furine site thereby forming an extracellular, highly glycosilated α
subunit, and a β subunit with an extracellular portion, that interacts with the ligand, a
transmembrane domain and an intracellular portion, containing a catalytic site. The
subunits are linked by a disolfure bond.
Met extracellular domain contains an homology region with the semaphorin, called
Sema domain, that includes α subunit and the N-terminus of the β chain, an affine-Met
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sequence rich in cysteines (MRS) followed by a motif made by repetition of prolyn-
glycin (G-P) and four structures immunoglobulin-like (Birchmeier et al 2003).
The intracellular domain contains three regions:
- a iuxtamembrane sequence with a Ser 985 that can be phosphorilated by
PKC or Ca2+
-calmoduline-dependent kinases in order to regulate receptor
activity (Gandino et al 1994)
- Tyr 1003 that can be bound by ubiquitin ligase Cbl, leading to
poliubiquitination, endocytosis and degradation of the receptor (Peschard
et al 2003).
- Tyrosine kinasic activity domain wich, following receptor activation, is
transphosphorilated on Tyr 1234 and 1235 and a C-terminal region that
contains Tyr 1349 e 1356 which are inserted into the multisubstrate
docking site, capable of recruiting downstream adapter proteins with Src
homology-2 (SH-2). These two Tyr are necessary and sufficient to
activate signal transduction both in vitro (Ponzetto et al.,1994) and in
vivo (Zanetti et al 1998).
Met ligand is HGF, or scatter factor (SF), secreted by fibroblasts in vitro or secreted by
platelets, as a potent epathic mitogenic agent, of patients suffering from acute epathic
insufficiency (Nakamura et al 1989; Zarnegar e Michaloppoulos 1989). Met expression
is usually related to epithelial cells, while HGF expression is restricted to mesenchymal
cells. Following stimulation with HGF, Met activation mediates several biologic effects
both in vivo and in vitro collectively known as “invasive growth”. In vivo invasive
growth HGF-induced involves cellular spreding, loss of cell-to-cell contact, acquisition
of a motile phenotype, also named scatter, that promotes cell migration towards a
different microenvironment. During this process cells lose their adherent junctions that
preserve their monolayer organization, change their polarization after cytoscheleton
rearrangements, digest ECM through proteases and dinamically remodel their adhesion
contacts to ECM. Finally, when cells reach the tissue, they proliferate and avoid
apoptotic mechanisms, eventually forming new vascular structures (Comoglio 2002).
After its activation, MET receptor autophsphorilates and binds to signal transducers and
adapting molecules involved in several molecular pathways that induce activation of
MAPK, PI3K, and Jak/STAT (Trusolino e Comoglio 2002). MET molecular pathway
alters expression and activity of cadherins, integrins and MMPs. These arrangements
cause loss of cell-to-cell contact, basal membrane disruption and alterates interaction
85
among components of the ECM, promoting tumour invasion across the stroma
(Trusolino e Comoglio 2002). Anyway HGF-dependent integrins regulation also allow
cancer cells to avoid anoikis (Longati et al 1996; Amicone et al 1997).
HGF-induced cell migration is related to increased expression/activity of SNAIL,
MAPK-EGR-1-mediated, with subsequent downregulation of E-cadherin expression,
while SNAIL expression alone in absence of HGF is not sufficient to induce a
migratory phenotype (Grotegut et al 2006). HGF-mediated signaling promotes
integrinic aggregation and recruitment of adhesive contacts and motile structures,
thereby leading to an increase in the percentage of integrins bound to actin cytoskeleton
inorder to form actin-rich cells protrusions (Trusolino et al 2000).
Met promoter positively responds to several stimuli growth factors and HGF-induced.
In addition, it has been demonstrated that several oncogenes like Ras and Ret induce its
overexpression (Engelman et al 2007; Ivan et al 1997). Met promoter analysis reveals
presence of four ETS sites, transcriptional factors that control genes involved inthe
invasive growth (Shirasaki et al 1999). For example, Ets1 promotes Met trancription in
vitro and is activated by Met itself through the MAPK pathway, suggesting a positive
feedback mechanisms that sustains invasive growth (Gambarotta et al 1996).
Another Met transcriptional regulator is tissutal O2 tension. In fact hypoxia upregulates
Met expression through HIF-1 binding to Met promoter, leading to amplification of
HGF signaling and invasive spur induction (Pennacchietti et al 2003).
Metastatic ability
Metastatization is promoted by tumour hypoxia and is related to a complex network of
events where chemokines direct cells migration, adhesion molecules mediate
colonization of distant organs and proteases digest/alterates ECM. Several proteins
involved in these processes include vimentin, fibronectin, keratins 14, 18, 19, MMPs,
cathepsin D, uPA/uPAr system are HIF-induced (Semenza 2003) (figura 24). Studies on
models of breast and renal cancer show that expression of chemokine receptor CXCR4,
major metastatic mediator, MMP-2 and 9 are all upregulated by HIF-1 (Leufgen et al
2005; Staller et al 2003).
Another key mediator of the metastatic process is the lysil oxidase enzyme, an HIF-1
target closely related to hypoxia and bad prognosis in several types of cancer. Lysil
oxidase modifies ECM components like elastyin and collagens and it has been observed
that its inhibition impairs in vitro migration and metastatization in vivo following
86
subcutaneous transplants and intravenous injection (Erler et al 2006). In addition, it has
been demonstrated that this family of enzymes promotes conformational changings in
nuclear factor SNAIL structure, promoting its resistence to degradation and repression
of E-cadherin expression, leading to EMT process (Pouyssegur et al 2006; Perinando et
al 2005).
According to the role of hypoxia in cancer cells survival, several studies show that HIF-
1α has an anti-apoptotic. In fact, HIF-1α downregulation promotes hypoxia-induced
apoptosis through caspases activation and inactivation of Bcl-XL, while HIF-1α
positively correlates with resistance to apoptosis in pancreatic cancer cells and hepatic
cancer cells HepG2 (Akakura et al 2001). In addition, a direct action by HIF-1α has
been hypothesized in pro-apoptotic Bid downregulation, as its promoter sequence
contains an HRE sequence (Earler et al 2004).
Genes transcription HIF-induced have a key role in tumour biology. For example Oct 4,
HIF-2α target, is a master regulator of both adult and embrional stem cells behavior (Tai
et al 2005). The mechanism through which Oct4 could modulate tumour growth has not
been elucidated yet, but it has been hypothesized that Oct-4 can promote cancer stem
cells development and growth, thereby promoting cancer self-renewal and resistence to
chemiotherapy.
HIFs also directly regulates transcription factors like c-Myc and Notch. In fact, HIF-1α
interacts with Notch-1 intracellular domain, thereby increasing its half-life and
transcriptional activity (Gustafsson et al 2005). The effect of this interaction in vivo has
not been cleared yet but could be important for tumour biology given the role of Notch
in mediating cell-cell communication, which involves gene regulation mechanisms that
control multiple cell differentiation processes during embryonic and adult life (Wilson e
Radtke 2006).
On the other hand, HIF-1α exerts an inhibitory effect on c-Myc. This inhibition
deregulates p21 and p27 (Koshiji M. et all 2004) and c-Myc target genes involved in
DNA “mismatch repair”, thereby suggesting a role of HIF in promoting hypoxia-
induced genetic instability (Koshiji et all 2005), while HIF-2α promotes c-Myc
transcriptional activity, promoting cancer progression (Gordan et al 2007).
miRNAs
It has been observed that HIF-1 not only regulates gene expression, but also activates
trascription of microRNAs (Fig. 25), a family of small, non coding, mRNAs made of
87
19-24 nucleotides, that influence stanility and translation of mRNAs (Kent e Mendell,
2006), thereby promoting cell surivival in the tumour microenvironment (Kulshreshtha
et al., 2007; Hebert et al., 2007).
In particular, HIF-1 induces expression of miRNA-210 (Kulshreshtha et al., 2007;
Camps et al., 2008; Fasanaro P et al., 2008), an ubiquitary hypoxia-responsive factor. In
several tumours miRNA-210 expression is increased in hypoxic areas rather than in
non-transformed tissues (Volinia et al., 2006; Iorio et al., 2005; Yanaihara et al., 2006;
Camps et al., 2008). In humans it has been pointed out that miRNA-210 is able to
regulate expression of genes involved in proliferation, DNA damage repairing systems,
chromatin remodelling, metabolism and cell migration (Kulshreshtha et al., 2007-a;
Camps et al., 2008; Fasanaro et al., 2008; Kulshreshtha et al., 2008; Kulshreshtha et al.,
2007-b).
Cellular metabolism
Aerobic energetic metabolism is dependent from oxidative phosphorilation and low O2
level severly impairs ATP production and cell survival. Whene there is lower O2
Fig. 25 Proposed model implicating select microRNAs in the hypoxia response.
88
availability, cells need to adapt reprogramming their metabolic behaviour. As
extensively described later, tumour cells undergo a “glycolitic switch” that allows them
to change their metabolism into an an anaerobic one, thereby keeping themselves
metabolically active even under lower O2 availabilty.
Physiologically non-neoplastic cells metabolize glucose into pyruvate, which is in turn
moved in the mitochondria, thereby entering the Krebs cycle and undergoing oxidative
phosphorilation. This metabolic pathway produces high ATP levels (38 molecules for
each glucose molecule), but needs O2 for mitochondrial respiration. On the other hand,
hypoxic cancer cells maintain high glycolitic activity but, in absence of oxygen, they
convert pyruvate into lactate, as high intracellular pyruvate concentration can severely
impair cell survival itself. Anyway, this metabolic changment alone is not sufficient in
order to satisfy cells metabolic needs as glycolisis leads to production of only two ATP
molecules, thuis cancer cells need other adaptive mechanisms.
Glycolisis metabolic products, mainly secreted lactic acid, induce a strong acidification
of the extracellular milieu, thereby lowering external pH level till 6.2-6.8 against
physiological values of 7.2-7.4 in normal tissues. This microenvironmental acidification
is direct consequence of increased glucose uptake and consumption, in order to sustain
cell vitality (Gatenby RA., Gillies RJ. 2004). In 1920 Otto Warburg first demonstrated
that cancer cells preferentially undergo glycolisis even in normoxic conditions. This
mechanism, thus named “Warburg effect”, allows cancer cells to take advantage on the
other not-transformed cell clones in hypoxic condition (see next chapter).
At the same time, cancer cells release pro-angiogenic factors that stimulates new vessels
formation in order to promote O2, glucose and other nutrients delivery to sustain
metabolic needs of highly proliferative cells. Also in this case hypoxia exerts a selective
pressure on cells unable to promote their angiogenic ability (Jiang et al 1997; Maxwell
et al 1997). In addition, hypoxia favours O2 delivery by erithrocytes promoting
expression og genes involved in erythropoiesis and Fe metabolism. There is an
increased EPO production, that stimulates red blood cells formation, thereby increasing
the number of circulating red blood cells (Semenza et al., 1991). In addition, in order to
ensure correct hemoglobin synthesis, as heme production is related to Fe availability,
there i san upregulation of genes involved in Fe metabolism. Hypoxia upregulates
expression of transferrin (Tf), that delivers Fe3+
in the bloodstream, of its receptor (Tfr),
which promotes Fe uptake, and of ceruloplasmin, which oxidizes ferrous Fe2+
to ferric
Fe3+
(Rolfs A et al., 1997; Bianchi L et al., 1999; Mukhopadhyay CK et al., 2000).
89
THE METABOLISM OF CANCER CELLS
Glycolisis
Glucose, a 6-carbons-aldheydic monosaccharide, is the primary source of energy for
plants, animals and most microorganisms. Its total oxidation in CO2 and H2O leads to a
free energy production of almost -2840 kJ/mole. Glicolisis is the key metabolic pathway
for glucose catabolism in most cells of the organism and it does not require O2 to carry
on its reactions.
The overall reaction of glycolysis is: a molecule of glucose (C6H12O6) reacts with 2
NAD+
molecules, 2 ADP molecules and 2 Pi molecules in order to produce 2 molecules
of NADH, 2 molecules of pyruvate (CH3COCOOH), 2 ATP molecules and 2 molecules
of H2O and 2 H+ ions.
1 glucose + 2 ADP + 2 Pi 2 pyruvates + 2 NADH + 2 ATP + 2 H2O + 2 H+
The overall molecular pathway is divided in ten reactions, The first five steps are
regarded as the preparatory phase, since they consume energy to convert the glucose
into two three-carbon sugar phosphates, while the second half of glycolysis is known as
the pay-off phase, characterised by a net gain of the energy-rich molecules ATP and
NADH (fig. 26).
During the preparatpry phase, each glucose molecule undergoes two phosphorilations
through consumation of two ATP molecules and is then divided into two molecules of
glyceraldehyde 3-phosphate. For every reaction there is a variation of standard free
energy G’0
(Gibbs energy) , defined as G
’0 = -RT lnK’eq where R is the gas constant
(R = 8,315 J/mole * K), T is the absolute temperature and K’eq is the equilibrium
constant. ∆G is the chemical potential that is minimized when a system reaches
equilibrium at constant pressure and temperature. Its derivative with respect to the
reaction coordinate of the system vanishes at the equilibrium point. As such, it is a
convenient criterion of spontaneity for processes with constant pressure and
temperature. Hence, out of this general natural tendency, a quantitative measure as to
how near or far a potential reaction is from this minimum is when the calculated
energetics of the process indicate that the change in Gibbs free energy ΔG is negative.
In essence, this means that such a reaction will be favoured and will release energy. The
energy released equals the maximum amount of work that can be performed as a result
of the chemical reaction. In contrast, if conditions indicated a positive ΔG, then
90
energy—in the form of work—would have to be added to the reacting system to make
the reaction go.
PREPARATORY PHASE REACTIONS :
1. Glucose + ATP Glucose 6-phosphate + ADP
G’0
=-17,7 kJ/mole
The first step in glycolysis is phosphorylation of glucose on the C-6 by a family of
enzymes called hexokinases to form glucose 6-phosphate (G6P). This enzyme uses
Mg2+
ions as cofactors and consumes ATP, but it acts in order to keep the glucose
concentration low, promoting continuous transport of glucose into the cell through the
plasma membrane transporters. In addition, it blocks the glucose from leaking out as the
cell lacks transporters for G6P, and free diffusion out of the cell is prevented due to the
charged nature of G6P. In addition, the disruption of the phosphoanidridic bound
between ATP phosphate groups generates high free energy variation which is partially
conserved in the formation of the phosphoesteric bound of G6P. finally, the interaction
between a phosphoric group and an enzymatic catalytic site contributes in lowering
activation energy, thereby promoting catalysis.
In animals, an isozyme of hexokinase called glucokinase is also used in the liver, which
has a much lower affinity for glucose (Km in the vicinity of normal glycemia), and
differs in regulatory properties. The different substrate affinity and alternate regulation
of this enzyme are a reflection of the role of the liver in maintaining blood sugar levels.
2. Glucose 6-phosphate Fructose 6-phosphate
G’0
= - 1,7 kJ/mole
G6P is then rearranged into fructose 6-phosphate (F6P) by glucose phosphate
isomerase, which uses Mg2+
ions as cofactors. Fructose can also enter the glycolytic
pathway by phosphorylation at this point.
The change in structure is an isomerization, in which the G6P has been converted to
F6P. The reaction requires an enzyme, phosphohexose isomerase, to proceed. This
reaction is freely reversible under normal cell conditions. However, it is often driven
forward because of a low concentration of F6P, which is constantly consumed during
91
the next step of glycolysis. Under conditions of high F6P concentration, this reaction
readily runs in reverse.
3. Fructose 6-phosphate + ATP Fructose 1,6-biphosphate + ADP
G’0
= -14,2 kJ/mole
Phosphofructokinase 1 (PFK-1) catalyzes the transfer of ATP phosphoric group to the
oxydrilic group of F6P. The reaction catalyzed by PFK-1, which requires Mg2+
ions as
cofactors, is coupled to the hydrolysis of ATP, an energetically favorable step, which
makes the reaction irreversible, and a different pathway must be used to do the reverse
conversion during gluconeogenesis. This makes the reaction a key regulatory point as
PFK-1 is positively regulated by AMP, ADP and fructose 2,6 biphosphate, catalytic
product of PFK-2 enzyme, concentrations, while it is negatively rergulated by high
concentrations of ATP and citrate, intermediate of the Krebs cycle.
Furthermore, the second phosphorylation event is necessary to allow the formation of
two charged groups in the subsequent step of glycolysis, ensuring the prevention of free
diffusion of substrates out of the cell.
The same reaction can also be catalyzed by pyrophosphate-dependent
phosphofructokinase (PPi-PFK), which is found in most plants, some bacteria, archea,
and protists, but not in animals. This enzyme uses pyrophosphate (PPi) as a phosphate
donor instead of ATP. It is a reversible reaction, increasing the flexibility of glycolytic
metabolism. A rarer ADP-dependent PFK enzyme variant has been identified in
archaean species.
4. Fructose 1,6-biphosphate Dihydroxyacetone phosphate + glyceraldehyde 3-
phosphate
G’0
= 23,8 kJ/mole
Destabilizing the molecule in the previous reaction allows the hexose ring to be split
by aldolase into two triose sugars, dihydroxyacetone phosphate, a ketone,
and glyceraldehyde 3-phosphate, an aldehyde. There are two classes of aldolases: class I
aldolases, present in animals and plants, and class II aldolases, present in fungi and
bacteria which use different mechanisms in cleaving the ketose ring.
92
Electrons delocalized in the carbon-carbon bond cleavage associate with the alcohol
group. The resulting carbanion is stabilized by the structure of the carbanion itself via
resonance charge distribution and by the presence of a charged ion prosthetic group.
The enzyme aldolase catalyzes an aldholic condensation that, despite the positive free
energy variation, can be reversible given the high intracellular concentrations of the two
reagents.
5. Dihydroxyacetone phosphate glyceraldehyde 3-phosphate
G’0
= 7,5 kJ/mole
Triosephosphate isomerase rapidly reversibly interconverts dihydroxyacetone phosphate
with glyceraldehyde 3-phosphate that proceeds further into glycolysis. This is
advantageous, as it directs dihydroxyacetone phosphate down the same pathway as
glyceraldehyde 3-phosphate, simplifying regulation.
PAY-OFF PHASE REACTIONS:
Since glucose leads to two triose sugars in the preparatory phase, each reaction in the
pay-off phase occurs twice per glucose molecule. This yields 2 NADH molecules and 4
ATP molecules, leading to a net gain of 2 NADH molecules and 2 ATP molecules from
the glycolytic pathway per glucose.
6. glyceraldehyde 3-phosphate + Pi + NAD+ 1,3-bisphosphoglycerate + NADH
+ H+
G’0
= 6,3 kJ/mole
The triose sugars are dehydrogenated and inorganic phosphate is added to them,
forming 1,3-bisphosphoglycerate.
The hydrogen is used to reduce two molecules of NAD+, a hydrogen carrier, to give
NADH + H+ for each triose.
Hydrogen atom balance and charge balance are both maintained because the phosphate
(Pi) group actually exists in the form of a hydrogen phosphate anion (HPO42-
), which
dissociates to contribute the extra H+ ion and gives a net charge of -3 on both sides.
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7. 1,3-bisphosphoglycerate + ADP 3-phosphoglycerate + ATP
G’0
= -18,5 kJ/mole
This step is the enzymatic transfer of a phosphate group from 1,3-
bisphosphoglycerate to ADP byphosphoglycerate kinase, forming ATP and 3-
phosphoglycerate. At this step, glycolysis has reached the break-even point: 2 molecules
of ATP were consumed, and 2 new molecules have now been synthesized. This step,
one of the two substrate-level phosphorylation steps, requires ADP; thus, when the cell
has plenty of ATP and little ADP, this reaction does not occur. Because ATP decays
relatively quickly when it is not metabolized, this is an important regulatory point in the
glycolytic pathway. Reactions 6 and 7 represent an energetic coupling where 1,3-BPG
is the common intermediate. Total reaction is favoured as the G’0
positive value of
reaction 6 is balanced by G’0
negative value of reaction 7.
ADP actually exists as ADPMg-, and ATP as ATPMg
2-, balancing the charges at -5 both
sides.
8. 3-phosphoglycerate 2-phosphoglycerate
G’0
= 4,4 kJ/mole
Phosphoglycerate mutase, which uses Mg2+
ions as cofactors,
now forms 2-
phosphoglycerate through placement of the phosphoric group from C-3 to C-2 of
glycerate. This is a two-phases reaction with generation of the intermediate of 2,3-
biphosphoglycerate.
9. 2-phosphoglycerate Phosphoenolpyruvate + H2O
G’0
= 7,5 kJ/mole
This reaction is catalyzed by enzyme enolase, which catalyzes the reversibile removal
of an H2O molecule from 2-phospholycerate thereby forming phosphoenolpyruvate
(PEP). Despite the little free energy variation, free energy derived from hydrolysis of
the phosphoric group increases.
94
10. Phosphoenolpyruvate + ADP Pyruvate + ATP
G’0
= -31,4 kJ/mole
This substrate-level phosphorylation is catalyzed by enzyme pyruvate kinase, which
requires K+ or Mg
2+ or Mn
2+ ions as cofactors. The reaction product is first in its enolic
form as enolpyruvate and then quickly tautomerizes in the chetonic form as pyruvate,
which is the commonest form at pH 7.
In aerobic conditions the NADH molecules are completely reoxydized by mitochondrial
complex I of the electron transport chain. The final electron acceptor is O2 which is then
reduced to H2O. in presence of O2, pyruvate is converted into acetyl-CoA by pyruvate
dehydrogenase complex, thereby entering the Krebs cycle.
Under low O2 tension, the pyruvate is reduced by lactate dehydrogenase to L-lactate in a
process called lactic acid fermentation, which is necessary for NAD+ regeneration, as in
hypoxic conditions NADH cannot be reoxydized. Loss of generation of NAD+ can be
harmful for cells because is the electrons acceptor required for oxydation of G3P and its
absence can block the glycolisis.
Pyruvate + NADH + H+ L-lactate + NAD
+ G
’0 = -25,1 kJ/mole
Lactate, produced for example in muscles undergoing high physical efforts, can be then
delivered to the liver and converted into glucose through gluconeogenesis. After that
glucose can be delivered again to muscles and catabolyzed through glycolisis. This
shunt between liver and muscles is also named Cori Cycle.
95
Fig. 26 Reactions of glycolisis.
96
The ” Warburg Effect”
In 1924 Otto Warburg first observed that cancer cells metabolyze higher amounts of
glucose in comparison with cells from normal tissues, producing high levels of lactate.
In addition, he showed that cancer cells preferentially have a glycolitic behaviour, even
in presence of high O2 tension (Warburg 1927). This peculiar characteristic was called
“aerobic glycolisis” or “Warburg Effect”. Further studies by Warburg itself
hypothesized that the Warburg effect was mainly related to a mitochondrial deficiency
developed by tumour cells, for example, through genetic mutations, thereby leading to a
low energy delivery from mitochondrial respiration. Finally, aerobic glycolisis had to be
the only possible metabolic mechanism that allowed cells to survive (Warburg 1956).
Anyway, subsequent studies demonstrated that mitochondrial deficiency was a rare
condition even in cancer cells (Weinhouse 1956). Today it has been clarified that
several factors, including tumour microenvironment and trascription factors, are needed
in order to sustain cancer progression.
In the case of metabolism, the choice by cancer cells to prefer a glycolitic behaviour
seems almost paradoxal because glycolisis is less efficient than mitochondrial
respiration in providing ATP. In fact, fermentative metabolism of a molecule of glucose
generates only 2 ATP molecules, while mitochondrial repiration leads to 36-38 ATP
molecules/glucose molecule.
The advantages coming from an aerobic glycolitic metabolism are still under
investigation. In multicellular organisms there are two main types of metabolism, the
one characteristic for highly proliferating and tumoural cells, and the quiescent
metabolism, typical of already differentiated cells. In order to divide quickly and
efficiently, highly proliferating cells need to replicate their genome, produce proteins,
lipids and all cellular components that are needed in order to form other cells. These
activities require an efficient delivery of nutrients from the extracellular space and their
conversion in biosinthetic products.
On the other hand, differentiated cells enter a quiescence phase (G0 phase) characterized
by high energetic demand in order to sustain cell vital. Thus cells convert glucose into
ATP and CO2 with the mitochondrial respiration in presence of high O2 tension, while
they catabolyze glucose into lactate just in hypoxic conditions.
An example of a proliferative cell energetic demand is the synthesis of palmytate, the
major component of cell membranes. Its production requires 7 ATP molecules, 16
atoms of carbon from 8 acetyl-CoA molecules and 28 electrons from 14 NADPH
97
molecules. Similarly, aminoacids and nucleotides synthesis requires more carbon atoms
and NADPH than ATP. A glucose molecule can generate 36 ATP molecules, or 30 ATP
molecules and 2 NADPH molecules if metabolyzed through the pentose phosphate
pathway. The same glucose molecule can also generate 6 atoms of carbon for the
synthesis of macromolecules but, in order to form a 16-atoms carbonic chain,
mitochondrial respiration needs 7 glucose molecules to produce the required amount of
NADPH. This high intermediates demand is just partially compensated by catabolism of
3 glucose molecules. In conclusion, it is clear that a highly proliferative cell, like the
tumoural one, can not use glucose just in order to produce ATP, otherwise the increased
ATP/ADP ratio would lower the glycolitic intermediate flux, thereby reducing acetyl-
CoA and NADPH production, required for macromolecules synthesis. A part of the
uptaken glucose must be delivered to production of macromolecules precursors like
acetyl-CoA for fatty acids, glycolitic intermediates for non essential aminoacids and
ribose for nucleotides. According to these observations, Increased ROS was
documented to modify a critical sulfhydryl group of pyruvate kinase M2 (PKM2),
rendering it inactive and resulting in the shunting of glucose away from glycolysis
toward the PPP (Anastasiou et al. 2011). The PPP generates NADPH, which reduces
glutathione into an active antioxidant that protects the cell. In this manner, the shunting
of glucose away from glycolysis toward the PPP is an essential element of redox
homeostasis.
In addition to oxidation of PKM2, increased ROS can stabilize HIF-1. HIF-1, in turn,
activates target genes such as PDK1, which diverts pyruvate away from mitochondrial
oxidation, and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4 (PFKFB4),
which degrades 2,6-fructose bisphosphate (2,6-FBP) (Keith et al. 2012; Semenza 2012).
2,6-FBP is a powerful allosteric activator of phosphofructose kinase 1 (PFK1), which
converts fructose-1-phosphate to fructose-1,6-bisphosphate (1,6-FBP) at a rate-limiting
step in glycolysis (Yalcin et al. 2009). Hence, increased PFKFB4, as observed in
prostate cancer cell lines, would diminish PFK1 activity and divert glucose into the PPP
shunt, elevating NAPDH to titrate ROS (Ros et al. 2012). It is notable, however, that
hypoxia also elevates PFKFB3, which drives glycolysis and can oppose PFKFB4; as
such, the balance between PFKFB3 and PFKFB4 activities is critical for shunting
glucose into glycolysis versus the PPP.
In addition, hypoxia adapting mechanisms involve coordinated expression of genes
coding for glucose transporters (GLUT1, GLUT3), glycolitic enzymes
98
(phosphogycerate kinase 1, pyruvate kinase M2, esokinase II), VEGF, EPO and heme-
oxygenase-1 (OH-1) (Semenza 2003). In addition, there is induction of pyruvate
dehydrogenase 1 (PDH-1) and lactate dehydrogenase (LDH) that reduce pyruvate
availability, thus increasing its convertion to lactate and inhibiting Krebs cycle
(Brahimi-Horn et al., 2007).
As a consequence, lowering of extracellular pH following lactic acid secretion acts as a
selective agent upon those cells unable to survive under acidic conditions. On the other
hand cells bearing low pH are able to produce ATP and alterate tumour
microenvironment, thereby directly harming the cells they compete with (Gatenby e
Gillies 2004). To this end HIF-1α upregulates expression of the monocarboxylate
transporter 4 (MTC4) that mediates lactic acid extrusion, and of the carbonic anidrase
IX (CA-IX), transmembrane enzyme that catalyzes convertion of extracellular CO2 into
carbonic acid (H2CO3). This mechanism further promotes extracellular acidification
through H+
ions release, while HCO3- intake allows cancer cells to avoid intracellular
acidification that could be highly disvital.
Finally, these data could at least partially explain the selective advantages of Warburg
metabolism.
99
Molecular pathways involved in cell metabolic regulation
PI3K. PI3K enzyme catalyzes conversion of phosphoinositides into phosphoinositides-
3,4,5-triphosphate (PIP3) (Cantley 2002). PI3K activity is physiologically regulated by
PTEN in non proliferative cells, but PTEN mutations can lead to a contistutive
activation of the PI3K pathway. According to that, there is a deregulated cell growth
and a continuative trasduction of survival signals that also affect cell metabolism. One
of the main PI3K effectors is Akt kinase and PIP3 binding to Akt pleckstrin homolgy
domain (PH domain) induces Akt translocation to the cell membrane, where it is
phosphorilated by PI3K-dependent kinase 1 and thereby activated (Bahskar et al. 2007).
Akt stimulates glycolisis and modulates ATP production through several mechanisms,
thereby allowing cancer cells to receive adequate bioenergetic supply. Akt induces a
glycolitic phenotype inducing GLUT expression on cell membrane.
In addition, Akt activates the glycolitic enzyme PFK-1. Akt phosphorilates and activates
PFK2 that in turn produces fructose 2,6-biphosphate, major PFK-1 allosteric activator
(Deprez et al. 1997). Finally, Akt can promote activation of complex 1 of the rapamycin
mammal target (mTOR). mTOR induces HIF-1 expression, leading to an
overexpression of almost all genes involved in cell glycolitic metabolism (Brugarolas
2003).
HIFs and MYC. As already described, hypoxia is a common features of solid tumours
and complexes HIF-1α, HIF-2α and HIF-3α are the master regulators of cell response to
hypoxia. In particular, HIF-1α regulates genes involved in cell metabolism (Hockel et
al. 2001).
HIF-1α can be activated in cancer cells in normoxic conditions. For example, mutations
that leads to PI3K costitutive activation also activates in an aberrant manner mTOR
pathway. In turn, mTOR phosphorilates ribosomal protein S6 kinase and binding
protein eIF-4E 1. Both these proteins induce an increased HIF-1 mRNA translation
(Semenza et al. 2001).
Recent studies have demonstrated that in models of hereditary paraganglioma,
glyoblastomas and renal carcinoma several oncosuppressor genes codificate for
enzymes of the Krebs cycle like succinate dehydrogenase (SDH), fumarate hydratase
(FH) and isocytrate dehydrogenase (IDH). SDH and FH loss of function leads to
accumulation of succinate and fumarate, respectively. Both these metabolites inhibits
100
PHD2 activity compiting with -ketoglutarate for binding its catalytic site. Thus PHD2
inhibition reduces hydroxylation, ubiquitination and proteasomal degradation of HIF-
1 (Selak et al. 2005; Isaacs et al. 2005; Koivunen et al. 2007; Hewitson et al. 2007).
Once activated, HIF-1α promotes gene transcription of SLC2A1 and SLC2A3, genes
coding for GLUT1, mainly expressed in erytrocytes, and GLUT3, mainly located in
neurons. Assocciation of these proteins create san hydrophilic channel for glucose
facilitated diffusion.
For what amplification of glycolitic pathway is concerned, HIF-1 induces expression of
esokinase 1 and 2 (HK1 e HK2), PFK1, aldolase A and C, glyceraldheyde-3-phosphate
dehydrogenase, phosphoglycerate kinase 1, enolase 1, pyruvate kinase and lactate
dehydrogenase A (LDHA).
In addition, recent studies on murine embrional fibroblasts (MEFs) have demonstrated
that gene coding for pyruvate dehydrogenase kinase 1 (PDK1) is a direct target of HIF-
1. This enzyme phosphorilates and inhibits the pyruvate dehydrogenase (PDH)
enzymatic complex, thereby blocking pyruvate entry into the Krebs cycle. These studies
suggest that suppressing Krebs cycle and mitochondrial respiration, PDK1 could act as
a survival factor for cells in condition of hypoxia (Kim JW et al. 2006).
c-Myc is a trtascription factor overexpressed in almost the 40% of human cancers. This
protein is a leucine-zipper HLH protein that dimerizes with the partner protein c-Myc-
associated protein X (MAX). c-Myc/MAX complex binds to DNA specific sequences
named E-boxes in order to activate or repress gene target transcription. The complex
target genes seems to be dependent from the cellular type, but there is a common group
of genes regulated by c-Myc like the ones involved in ribosomal biogenesis and glucose
metabolism (Dang et al. 2008). It has been observed that c-Myc activates LDHA
expression, thereby increasing lactate production in a model of lung carcinoma (Dang et
al. 1997), while further studies suggest that also aldolase A, enolase, esokinase 2,
GAPDH, GPI, HK2 PFK1, PGK, PGM e TPI are upregolated by c-Myc activation
(Osthus et al. 2000).
AMPK. Protein kinase activated by AMP (AMPK) is the main sensor of intracellular
energetic state and has a central role in metabolic stress response. AMPK is activated
through phosphorilation from protein kinase LKB1. In a condition of metabolic stress
AMPK is activated by an increased AMP/ATP ratio. AMPK acts on glycolisis
phosphorilating and activating the PFK2 inducible isoform (PFKFB3) (Marsin et al.
2000). PFK2 reaction product, fructose 2,6-biphosphate, is a PFK1 activator.
101
Observations from in vivo models lacking LKB1 and AMPK show a costitutive
activation of the mTOR pathway. In the same conditions it has been observed an
upregulation of HIF-1 and its target genes in normoxic condition (Shackelford et al.
2009). These data show a strong dependence between LKB1/AMPK loss and HIF-1
expression through an mTOR-dependent pathway.
p53 and OCT1. p53 is a transcription factor and oncosuppressor gene that has a major
role in cell response to conditions of stress. It has been observed that p53 induces
exokinase 2 expression through direct binding to the promoter, thereby stimulationg
glucose 6-phosphate production (Mathupala et al. 1997).
p53 regulates cellular glucose metabolism through activation of two effectors: TIGAR
and SCO2. TIGAR is a p53-inducible gene and has a high functional affinity for PFK2
phosphatasic domain (Bensaad et al. 2006). PFK2 is a bifunctional enzyme
characterized by a kinasic domain with high affinity for fructose 6-phosphate, and a
phosphatasic domain with high affinity for fructose 2, 6 biphosphate. Thus TIGAR
expression induces fructose 6-phosphate production in the glycolitic pathway and its
conversion to glucose 6-phosphate, according to the law of mass action. Finally,
glucose 6 phosphate can enter the pentose phosphate pathway in order to produce
NADPH and ribose-5-phosphate (R5P) for nucelic acids sinthesis.
Second effector gene SCO2 is directly activated after binding to p53 as shown in
models of colon cancer (Matoba et al. 2006). Together with SCO1, SCO2 encodes for
two subunits of cytocrome c oxydase (COX), which catalyzes translocation of reducing
equivalents from cytocrome c to O2, pumping protons through the inner mitochondrial
membrane.
Finally, it has been demonstrated that the promoter sequence of the gene coding PTEN
contains a p53 binding region, through which p53 induces PTEN mRNA expression and
thereby PI3K pathway inactivation (Stambolic et al. 2000).
OCT1 is a transcription factor from the POU domain family (Pit-1, Oct1/2, Unc-86).
Studies on MEFs and adenocarcinoma epithelial cells have showed that OCT1 loss of
function induces a metabolic changment that suppresses tumour progression. In
particular, OCT1-/-
cells show a decreased of glucose and lactate accumulation and an
increased concentration of Krebs ciycle intermediates like malate, succinate and
isocytrate. These results demonstrate an increased mitochondrial activity and a
decreased glycolitic flux due to OCT1 loss (Shakya et al. 2009).
102
Fig. 27 Regulation of metabolic pathways by HIF-1, MYC, Oct-1 and p53.
Pyruvate Kinase M2 (PKM2). Pyruvate kinase (PK) catalyzes conversion of PEP to
pyruvate with substrate-level phosphorilation of ADP to ATP.
There are several PK isoforms that are expressed according to specific metabolic needs
of the tissue. PK L is expressed in tissues with high gluconeogenic activity like liver
and kidenys, while isoform R is mainly expressed in erithrocytes and PK-M1 is located
in tissue with high ATP demand like heart and brain. Isoform M2 (PKM2) is mainly
expressed in lungs and, in general, by cells with high nucleic acids syntethic activity
like embrional cells, adult stem cells and cancer cells. PK L and R are coded from the
same gene, PKLR, but are under control of two different promoters. Isoenzymes PKM1
and PKM2 are different splicing products from the same mRNA of gene PKM2 and
differ for 21 aminoacids (Reinacher et al 1981; Eigenbrodt et al. 1985; Eigenbrodt et al.
1992; Eigenbrodt et al. 1986; Steinberg et al. 1999). PKM2 transcription is activated by
transcription factor Sp1, while transcription factor Sp3 acts in hypoxic conditions
repressing its transcription after physical dissociation from the promoter.
Immunohystochemistry studies of PKM2 have demonstrated that this protein can
associate in a quaternari structure in a different manner in lung and cancer cells (fig.
28). In lungs and healthy proliferative cells PKM2 is expressed in a tetrameric form
with four identical subunits, while in cancer cells, is always expressed in a dimeric
form, and this particular PKM2 structure is also named “tumoural PKM2”. Tetrameric
form PKM2 shows higher affinity for PEP, while dimeric form is caharacterized by
103
lower activity. In presence of PEP physiological concentrations, tetrameric PKM2 is
highly active, while dimeric form is almost totally inactive. This catalytical difference is
amplified when tetrameric form associates with glycolitic enzymes like HK, G3PDH,
PGK, PGM, enolase and LDH-A and other enzymes like nucleotide diphosphate kinase,
adenilate cyclase, glucose-6-phosphate dehydrogenase and proteins of the
RAF/MEK/ERK pathway. On the other hand, dimeric PKM2 do not associates to
glycolitic enzymes. This complex has two main functions: compartimentalization of
metabolic reactions into the cytosol and increasing glycolisis efficiency through binding
to enxymes involved (Eigenbrodt et al. 2005). Transition from tetrameric to dimeric
form is induced after direct interaction between PKM2 and several oncogenes
(Eigenbrodt et al. 1988).
Fig. 28 PKM2 molecular structure.
Recent studies have clarified the molecular mechanism of M1- o M2- splicing of PKM2
mRNA. In particular, it has been demonstrated that heterogenous ribonuclear proteins
(hnRNP) I, A1 and A2 bind to RNA sequence on exon 9 inhibiting M1-specific splicing
and c-Myc activates transcription of hnRNPI, hnRNPA1 and hnRNPA2. Thus it is clear
that tumour cells with constitutive c-Myc expression have higher PKM2 basal
expression levels (Chen et al. 2010; Clower et al. 2010).
PK inactive isoform expression could represent an advantage for proliferating and
tumour cells as, blocking glycolisis, carbonic flux stops at PEP formation. For the law
of mass action, accumulation of this metabolite reverts the reaction flux, thereby leading
glycolitic intermediates to biosintethic pathways, like the pentose phosphate pathway.
These reactions are necessary to cancer cells as they provide both ribose-5-phosphate
104
for nucleotides synthesis and reducing power with NADPH, thereby avoiding axcess of
ROS and conditions of potentially harmful oxidative stress.
According to that, several mechanisms of PKM2 enzymatic activity inhibition have
been described, like oxidation, phosphorilation and acetylation. Lv and collegues have
demonstrated in a prostatic carcinoma model that PKM2 acetylation on K305, induced
by high glucose concentration, impairs its catalytical activity. In fact, acetylation
favours PKM2 interaction with chaperon HSC70 and susequent association with
lysosomes, thereby leading to a mechanism of chaperone-mediated autophagy (CMA).
Thus cancer cells accumulates glyoclitic intermediates, thereby activating anabolic
pathways and promoting proliferation and tumour growth in in vivo models (Lv et al.
2011). In addition, it has been recently showed in several cancer models, like breast and
lung carcinomas, that PKM2 is phosphorilated on Tyr Y105 by FGFR1. This post-
translational modification promotes dissociation of PKM2 tetrameric complex from its
cofactor fructose 1, 6 biphosphate, impairing its activity (Hitosugi et al. 2010).
Finally, also ROS seem to have a key role in regulating PKM2. In a model of lung
carcinoma it has been demonstrated that acute increase of intracellular ROS oxydizes
the Cys358 of PKM2 catalytic site, modifying its structure and reducing catalysis. This
event is strictly related to glucose catabolism through the pentose phosphate pathway,
thereby leading NADPH synthesis in order to buffer excess of ROS. Cys358 oxidation
is crucial because its mutation in a Ser358 blocks this event, thereby leading cells to
higher sensitivity to oxidative stress and reducing their survival and tumour growth in
vivo (Anastasiou et al. 2011). According to this data, Anastasiou and collegues have
recently demonstrated that both PKM1 overexpression and stimulation with
pharmacological small-molecule PKM2 activators inhibits the growth of xenograft
tumours. These small-molecule activators bind PKM2 and promote a constitutively
active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins,
thereby leading to direct interference with anabolic metabolism (Anastasiou et al. 2012).
In addition, PKM2 has a role as direct coactivator of HIF-1α in target genes
transcription (fig. 29).
As previously stated, PHD2 has a central role in hydroxilating and then promoting HIF-
1α degradation. For what concerns PHD3, recent studies have demonstrated that PHD3
hydroxilates prolines residues inside a highly conserved motif LRRLAP on the target
protein (Xie et al. 2009). LRRLAP has been identified in an internal sequence of exon
10 of PKM2 gene. In particular, Pro403 and Pro408 hydroxylation on PKM2 by PHD3
105
positively correlates with expression of HIF-1α main target genes and probably
promotes PKM2 transition from tetrameric to dimeric form (Chen et al. 2011). PKM2
dimeric form migrates in the nucleus, interacts with HIF-1α through direct binding to
HREs sequences on promoters of target genes and recruiting HIF coactivator p300. On
the other hand, PKM1 do not associates to HIF-1α (Semenza et al 2011). According to
that, there is an increased transcription of HIF target genes like PKM2 and EGLN3,
encoding forr PKM2 and PHD3, respectively (Pescador et al. 2005). These observations
suggest a positive feedback mechanism of HIF-1α activation in order to promote
metabolic reprogramming of cancer cells (Semenza et al. 2011).
Fig. 29 Role of PKM2 as HIF-1α direct coactivator.
106
Models of metabolic symbiosis
Nutrients and O2 delivery to tumour mass is mediated by angiogenesis, but new
vascularization is often made by arterious vessels not connected to venous circulation
(Fukumura et al. 2010). In addition, tumour burden is characterized by impaired
perfusion if compared to healthy tissues, thereby leading to formation of hypoxic areas
inside the tumour, with subsequent HIF-1α stabilization (Le et al. 2010).
This event leads cells to exploit different metabolic mechanisms inside the same tumour
mass: normoxic regions, close to blood vessels, preferentially exploit mitochondrial
respiration and phosphorilative oxidation, while hypoxic cells undergo glycolisis.
According to that, it has been demonstrated that lactate, glycolisis end product, is the
key mediator of a “metabolic symbiosis” model where both glycolitic and respiring
cells reciprocally regulate nutrients availability through the transporter MCT-1. In fact,
its inhibition shows antitunoral effects (Sonveaux et al. 2008).
According to this model, normoxic subpopulation of cancer cells exploit extracellular
lactate to fuel oxidative phosphorilation, thereby avoiding extracellular glucose
depletivo, as glucose is metabolyzed through glycolisis by hypoxic cancer cells. Lactate
used for mitochondrial metabolism gives several advantages to cancer cells:
pyruvate oxidation to lactate by lactate dehydrogenase leads to production of
reducing power that prevents harmful intracellular oxidative stress, thereby
favouring cells survival (Lee et al. 2003; Pelicano et al. 2006);
lactate oxidation do not requires ATP consumption;
each molecule of lactate generates 18 ATP molecules, thereby allowing cells to
save energy for glycolitic enzymes activity.
Lactate delivery between hypoxic and normoxic tumour areas is similar to the
physiologic mechanism of muscles (Halestrap et al. 2004; Halestrap et al. 1999). During
intende physical efforts, white mucle fibres produce and release lactic acid
(Dubouchaud et al. 2000). Extracellular lactate is then uptaken by red muscle fibres in
order to promote oxidative metabolism (Baker et al. 1998; Bonen et al. 1998;
Dubouchaud et al. 2000) and thuis process is mediated by both MCT-1 and MCT-4
transporters (Halestrap et al. 2004; Halestrap et al. 1999). MCT-4 has low affinity for
lactate (Km=22 mM) and is consequently expressed by glycolitic cells in order to
release the metabolite. MCT-4 expression is HIF-1α-dependent (Dimmer et al. 2000;
Ullah et al. 2006).
107
On the other hand, MCT-1 has high affinity for lactate (Km=3,5-10 mM), thereby
promoting its uptake in respiring cells (Halestrap et al. 1999; Dubouchaud et al. 2000;
Ullah et al. 2006). It has been demonstrated that MCT-1 is the main canne for lactate
intake by cancer cells (Sonveaux et al. 2008), while MCT-4 is responsible for protons
extrusion from glycolitic cancer cells (Wahl et al. 2002). According to that, MCT-1
gene silencing is sufficient to impair oxidative phosphorilation lactate-dependent and
cancer cells survival (Sonveaux et al. 2008). Despite being a pharmacological target to
kill cancer cells close to blood vessels, MCT-1 inhibition undirectly promotes necrosis
in hypoxic cancer cells usually resistent to conventional teraphies and causing cancer
recidival (Brown et al. 2004) (fig. 30). Finally, cancer cells death for lack of glucose is
caused by metabolic modifications of respiring cells. In fact, oxidative cells modify
their metabolic behavior to a glycolitic one, caused by inhibition of cellular respiration
(Crabtree effect) (Crabtree 1929).
The lactate/MCT-1 pathway represents the key event of metabolic symbiosis in cancer
and its discovery leads to new interpretations of data and relations previously observed
(Sonveaux et al. 2008). For example, high energetic nature of lactate explains its
autocrine activity as growth factor (Pike et al. 1991) and its bidirectional delivery
among different cell types (Spencer et al.1976; Cheeti et al. 2006; Wang et al. 2007). In
addition, seveal studies relate to neoplastic cells a mechanism described just for stromal
cells in order to exploit cancer cells metabolic products (Sonveaux et al. 2008).
Inhibition of MCT-1 impairs normoxic cancer cells ability to uptake lactate for their
oxidative phosphorilation. Subsequently this cancer cells subpopulation exploits
extracellular glucose for its metabolic needs, thereby depleting glucose from hypoxic
cells that are unable to survive.
A second effect of MCT-1 inhibition, at least in vitro, is the lethal intracellular
acidification (Wahl et al. 2002; Belt et al. 1979; Coss et al. 2003; Fang et al. 2006;
Mathupala et al. 2004). In fact, it has been observed that MCT-1 expression is increased
also in cancer cells that metabolyze butirrate, as this tranporter promotes its intake
(Serpa et al. 2011). Butirrate is used by cancer cells to fuel β-oxidation, as demonstrated
by their overexpression of acyl-dehydrogenase enzyme. Thus cancer cells able to
metabolize butirrate are positively selected by tumour microenvrionment (Serpa et al.
2010). These cells show a mesenchymal phenotype, with E-cadherin downregulation,
increased expression of MMP-2 and 9 e and expression of α2 and α3 integrins.
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Fig. 30 Model of metabolic symbiosis between hypoxic and respiring cells.
In addition, recent studies suggest that tumour microenvironment, primarily cancer-
associated fibroblast (CAFs), has a role in cancer metabolism. These observations lead
to models according to which CAFs fuel neoplastic cells with aminoacids and
nucleotides through autophagy, thereby promoting cancer progression and
metastatization (Pavlides et al. 2010). In particular, several studies have focused on
expression analysis of protein caveolin-1 1 (Cav-1) on CAFs. Cav-1 belongs to the
caveolins transmembrane proteins family, main components of membrane
microdomains called caveolae and involved in mechanisms of endocytosis receptor-
independent. Cav-1 is mainly expressed on endothelial cells, adipose and stromal cells.
In aprticular, Lisanti and collegues have demonstrated that low Cav-1 expression on
CAFs extracellular membrane is correlated to a highly proliferative and metastatic
phenotype in a model of breast cancer (Migneco et al. 2010). Compairing this model to
the relation between hypoxic and normoxic cancer cells, supported by an O2 perfusion
gradient inside the tumour, the metabolic relation between cancer cells and CAFs is
mainly driven by a condition of high oxidative stress. According to that, correlation
between oxidative stress and Cav-1 loss shows a biunivocal feature: Cav-1 degradation
induces an increase in ROS accumulation and, in turn, oxidative stress induced by
109
neighbouring cancer cells further promotes Cav-1 degradation. Cav-1 has a role as
negative regulator NO production through inhibition of NO syntase. Loss of Cav-1
thereby promotes increase in NO production, which can inhibit mitochondrial functions
acting a san inhibitor of ferrous protein of the electron chain transport. This inhibition of
oxidative phosphorilation NO-mediated induces higher mitochondrial ROS production
in addition, studies on coc-colture models between fibroblasto and MCF-7 breast cancer
cells showed that oxidative stress cancer cells-induced leads to Cav-1 digestion by
fibroblasts and this effect is highly reduced after administration of antioxidant
molecules like N-acetyl cystein (NAC) (Bonuccelli et al. 2010). Other studies
hypothesized that ROS act on PHDs, inactivating them and promoting HIF-1α
stabolization.
It has been clearly demonstrated that phosphorilation or other post-tradutional
modifications ROS-dependent on PHDs induce modifications on their catalytic activity
(Klimova et al. 2008). Subsequently fibroblasts exposed to oxidative stress undergo a
metabolic shift towards glycolisis ROS and HIF-1α dependent and produce and estrude
high amounts of lactate through MCT-4. Excreted lactate is then taken up by cancer
cells through MCT-1 in order to enter the Krebs cycle. This new metabolic model is
also named “reverse Warburg effect” (Martinez-Outschoorn et al. 2010).
This metabolic model has been recently confirmed also in a prostatic carcinoma model.
Studies conducted by our research group have demonstrated that, analyzing expression
patterns of metabolic genes from both fibroblasts from benign hyperplasias and CAFs,
the latter are characterized by a metabolic reprogramming to a Warburg phenotype and
by a condition of mitochondrial oxidative stress. This event is caused by interaction
with prostatic cancer cells that promotes fibroblasto activation, GLUT-1 expression,
lactate production and extrusion after de novo expression of MCT-4. These metabolic
modifications are HIF-1α-dependent, expressedd in normoxic condition following
reduction of SIRT-3 expression. SIRT-3 is a NAD-dependent protein that, buffe ring
excess of ROS, promotes PHDs activity and HIF1α downregulation. Subsequently, lack
of this protein expression leads to accumulation of ROS, impaired PHDs activity and
HIF-1α increased expression.
At the same time, cancer cells respond to CAFs contact modifying their metabolism to a
respiring metabolism, reducing GLUT-1 expression and thereby increasing lactate
intake through de novo expression of MCT-1. Thus cancer cells progressively become
independent from glucose consumption, thereby developing high dependance from
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extracellular lactate intake in order to promote anabolic reactions. According to these
observations, MCT-1 pharmacological inhibition with 4-cyanohydroxycinnamide
(CHC), blocking lactate influx, strongly reduces cancer cells survival and growth.
In conclusion, taking advantage from modyfing the surrounding microenvironment,
cancer cells create a strong symbiotic interrelationship with CAFs, thereby surviving in
presence of low glucose availability (Fiaschi et al. 2012) (fig. 31).
Fig. 31 Model of metabolic symbiosis between cancer cells and cancer-associated fibroblasts
(CAFs).
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MATERIALS AND METHODS
MATERIALS
Unless specified, all reagents used for cell cultures were purchased from
Euroclone Group, except DMEM 1x (no glucose) from Invitrogen, pyruvate and
lactate from Sigma-Aldrich.
Noradrenaline, adrenaline and propanolol were from Sigma-Aldrich.
Cytokines used for cell stimulation were purchased from Peprotech.
The metalloproteinase farmacological inhibitor Ilomastat was purchased from
Chemicon International.
The HIF-1α farmacological inhibitor Topotecan was purchased from Sigma-
Aldrich.
Transwells for invasion assays were from Euroclone Group.
The Diff-Quik staining kit was purchased from BIOMAP SNC.
Matrigel was purchased from BD Biosciences.
Proteases and phosphateses inhibitors were from Sigma-Aldrich.
Bradford reagent for protein dosage and all materials for SDS-PAGE were from
Biorad.
PVDF membrane (Polyvinylidene fluoride) used for western blotting was from
Millipore.
All the antibodies used were purchased from Santa Cruz Biotechnology except
antibodies against α-SMA (Sigma-Aldrich), HIF-1α (BD Biosciences),
p38/phospho-p38 and p42-p44 MAPK/phospho-p42-p44 MAPK (Cell
Signaling).
112
The secondary antibodies enzyme horseradish peroxidase –coniugated HRP)
were from GE Health Care.
Chemiluminescence revelation kit is from GE Health Care.
The photographic plates were from Kodak.
The metalloproteinase catalytic activity evaluation kit AmpliteTM
Universal
Fluorimetric MMP Activity Assay Kit - Red Fluorescence was purchased from
AAT Bioquest.
The cytofluorimetric apoptosis staining kit Annexin V-IP Fluos Staining Kit was
from Roche.
Common use solutions
SDS−PAGE 1X Sample Buffer. For 100 ml: 0.01 g of Bromophenol Blue, 1.52
g Tris Base, 20 ml glycerol, 2 g di SDS, 20 ml di 2-mercaptoethanol.
SDS-PAGE 1X running buffer. For 1 litre: 25 mM Tris, 192 mM glycin, 0.1%
(W/V) SDS, pH 8.3.
SDS-PAGE 1X blotting buffer. For 1 litre: 25 mM Tris, 192 mM glycin, pH 8.3,
10% methanol.
RIPA lysis buffer (50 mM Tris.HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2
mM EGTA, 1mM sodium ortovanadate, 100 mM NaF).
Washing solution: tween 0.1 % in PBS.
Blocking solution: non-fat dry milk 2 %, tween 0.05 % in PBS.
Stripping solution: 62.5 mM Tris Hcl pH 6.8, 2 % SDS, 100 mM β-
mercaptoethanol.
PBS (Phosphate buffered saline). For 1 litre: 0.2 g di KH2PO4, 0.2 g KCl, 0.8 g
NaCl , NaH2PO4 pH 7.4.
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METHODS
Cell cultures and treatments
Human dermal fibroblasts (HDFs), human A375 cells isolated from amelanocytic
melanoma and human Hs29-4T cells isolated from human melanoma lymphonodal
metastasis were all cultivated in DMEM high glucose (4,5 g/L) (Dulbecco’s Modified
Essential Medium), supplemented with 10% bovine fetal serum (FCS), glutammine 2
mM, penicillin (100 U/ml) and streptomicin (100 μg/ml), at 37°C in a 5% CO2
humidified atmosphere. Cells are usually stored in liquid nitrogen in a freezing solution
containing 90% FCS and 10% dimethyl sulfoxide (DMSO) and then plated in Petri
capsules.
All experiments were performed with 70%-80% confluent cultures, following 18-h
incubation in serum-free culture medium. Cells were then stimulated with NE or E, at
1µM concentration. Where needed, cells were pre-treated with unselective β-AR
antagonist propranolol (Sigma-Aldrich) (1 µM). After 1h, medium was removed and
cells were stimulated with NE 1 µM with or without propranolol 1 µM.
For metabolism experiments, 70%-80% confluent A375 cells were incubated in
normoxic condition for 18 h in serum-free and nutrient-free culture medium DMEM 1X
w/o glucose/pyruvate/HEPES. Cells were than cultured for 24 h in presence of glucose
(4,5 g/L), pyruvate (10 mM) and lactate (10 mM), in presence or absence of serum.
Isolation of human dermal fibroblasts
Human dermal fibroblasts (HDFs) were isolated from a surgical explantation taken from
healthy patients. A small slice of the tissue piece was minced with sterile scalpels and
pieces of <1 mm in size were plated in Petri plates and covered with covering glasses,
favouring pieces compression and fragmentation. After having been digested overnight
in 1 mg/mL collagenase I at 37 C°, fragments were spun down, re-suspended and plated
in complete DMEM supplemented with 20% FCS, glutammine 2 mM, penicillin (100
U/ml) and streptomicin (100 μg/ml), Kanamicyn and AMFO (Amphotericyn B) 100
mg/lt.
After 3-4 days culture medium was removed and fresh medium was then added. Days
required for epithelial and fibroblast cells exit from bioptic fragments is highly variable
114
and depends on the type of bioptic tissue and the number of fibroblasts composing it.
Usually it takes 20-30 days and, after this period, tissutal fragments were removed with
steril tweezers and trypsinyzed, thereby promoting fibroblasts isolation as epithelial
cells are not able to re-adhere to plate surface. Obtained fibroblasts were then
maintained in culture with complete DMEM supplemented with 10% FCS, glutammine
2 mM, penicillin (100 U/ml) and streptomicin (100 μg/ml). We also excluded
contamination by skin stem cells of HDFs by evaluation of CD34 and cytokeratin-15,
acknowledged as skin stem cell markers.
Preparation of conditioned media
For preparation of conditioned media, A375 cells were grown to 70% confluence in
complete medium for 24 h. Then medium was removed and, after washing in PBS
solution, cells were serum starved overnight in order to promote cells entry into a
quiescent G0 phase, thereby better evaluating cells responsiveness to exogenous
treatments. After this period cells were treated for 24 h with the indicated cytokines:
TGF-β 10 ng/ml, NE 1 µM, VEGF (20 µg/ml) and IL-6 (50 µg/ml) added to serum-free
culture medium.
Fresh serum-free medium was added for an additional 24 h before collection of
conditioned medium (CM), in order to obtain CM free from cytokines (but conditioned
by their earlier administration). CM were then harvested, clarified by centrifugation, and
used freshly on HDFs cells that were then incubated with the obtained CM for 24 h and
used for Western blot analyses.
Cell lysis and protein quantification
In order to identify proteins of interest, cell lysates were then separated through SDS-
PAGE (polyacrylammide gel elettrophoresis) and revealed with Western Blotting
analysis.
After stimulations, cells were washed once with PBS solution and then lysated with
RIPA lysis buffer. Obtained protein lysates were collected, always kept in ice and
centrifugated at 13000 rpm for 15 minutes. After centrifugation, supernatant was
collected and total proteins were quantified with Bradford assay.
115
Total protein quantification, espresse in g/ml, is evaluated with Coomassie Brilliant
Blue, which binds to basics and aromatics aminoacidic residues (specially arginins),
leading to maximum absorption at 595 nm wavelegth. Thus, Coomassie Brilliant Blue
intensity is positively correlated to protein concentration.
For the standard curve bovine serum albumine was used (BSA), diluting BSA 2 mg/ml
concentrated in deionized water and then obtaining rising BSA concentrations from 2
g/ml to 15 g/ml.
Then Bradford reagent is prepared diluting 1/5 of starting solution with Coomassie
Brilliant Blue in 4/5 of deionized water.
To run the assay, 50 l of each sample, opportunely diluted in water in labelled
eppendorfs, must be added to 950 l of the working solution for each sample,
resuspending well. After a 5 minutes incubation, the absorbance of each sample is
evaluated at a wavelength of 595 nm, subtracting the blank value. From values obtained
from the standard curve it is possible to create a curve of absorbance in function of its
concentration, thus, interpolating absorbances values to the standard curve, it is possible
to calculate final protein concentration.
Correlation between absorbance and concentration is expressed by Lambert-Beer law: A
= dc, where represents the extinction coefficient, d the path length and c represents
sample concentration.
For each Western Blotting experiment usually 20-25 g of total proteins are loaded on
SDS-PAGE for each sample.
SDS-PAGE analysis
Sodium dodecyl sulfate polyacrylammide gel electrophoresis (SDS-PAGE) is a
technique for separating proteins based on their ability to move within an electrical
current, which is a function of the length of their polypeptide chains or of their
molecular weight. This is achieved by adding SDS detergent to remove secondary and
tertiary protein structures and to maintain the proteins as polypeptide chains. The
anionic SDS coats the proteins (almost one SDS molecule binds every two aminoacidic
residues of the polipeptidic chain), mostly proportional to their molecular weight, and
confers the same negative electrical charge across all proteins in the sample.
SDS-PAGE samples are boiled for 5 minutes in a sample buffer containing SDS and -
mercaptoethanol, which leads to disolfuric bonds reduction and destabilization of
116
eventual protein tertiary structure. In addition, sample buffer is supplemented with
bromophenol blue, ionizing coloured-tracking solution for the electrophoretic run, and
glycerol, which increases sample density and promotes its stratification ate the bottom
of the loading well.
Once finished samples loading in the stacking gel, an electric field is applied across the
gel, causing the negatively-charged proteins to migrate across the gel towards the
positive electrode (anode). Stacking gel, characterized by very low acrilamide
concentration (4%), is required to better stratificate the samples before entering the
separating gel. According to that, different ionic force and pH of running buffer and
stacking gel are required: this technique is called istachophoresis. In isotachophoresis
the sample is introduced between a fast leading electrolyte and a slow terminating
electrolyte. After application of an electric potential a low electrical field is created in
the leading electrolyte and a high electrical field in the terminating electrolyte. The pH
at sample level is determined by the counter-ion of the leading electrolyte that migrates
in the opposite direction. In the first stage the sample constituents migrate at different
speeds and start to separate from each other. The faster constituents will create a lower
electrical field in the leading part of the sample zone and viceversa. Finally the
constituents will completely separate from each other and concentrate at an equilibrium
concentration, surrounded by sharp electrical field differences. Specific spacer or
marker molecules are added to the sample to physically separate the sample constituents
one is interested in.
Separation of SDS-proteins complexes is achieved according to separating gel
acrylamide concentration. Lower percentage gels are better for resolving very high
molecular weight proteins, while much higher percentages are needed to resolve smaller
proteins, while bromophenol blue is a very small molecule which is not affected by
fricitional forces, thereby representing the migration front. Proteins relative molecular
mass is evaluated by comparison with protein ladder standard molecular weights,
separated in the same gel. Running is carried on at 200 Volts for almost 1 h.
Western Blotting
After run, polyacrylamide gel is maintained for 5 minutes at room temperature in slow
agitatation in the transfer blot. In order to make the proteins accessible to antibody
detection they are moved from within the gel onto a membrane made of polyvinylidene
117
difluoride (PVDF). The method for transferring the proteins is called electroblotting and
uses an electric current to pull proteins from the gel into the PVDF membrane. The
proteins move from within the gel onto the membrane while maintaining the
organization they had within the gel. Proteins transfer is carried out at 100 Volts for 1 h.
PVDF membrane must be previously activated through treatment with methanol for 15
seconds and left at drying for at least 15 minutes at room temperature.
After electroblotting the PVDF membrane is incubated 1 h in slow agitation at room
temperature with specific primary antibodies in a blocking solution containing non-fat
dry milk 2% and Tween 0.05%. After incubation, the membrane is washed three times
with a washing solution containing PBS 1X and Tween 0.1% and, in order to reveal the
specific protein, the membrane is incubated with horseradish peroxidase conjugated
secondary antibody for 30 minutes and then washed again for three times. In the
chemiluminescence reaction horseradish peroxidase catalyzes the oxidation of luminol
into a reagent which emits light when it decays. Since the oxidation of luminol is
catalyzed by horseradish peroxidase, and the HRP is complexed with the protein of
interest on the membrane, the amount and location of light that HRP catalyzes the
emission of, is directly correlated with the location and amount of protein on the
membrane. Chemiluminescent protein revelation is carried out with ECL-Amersham
Pharmacia kit reagents and developing of blots is carried out in the developing room
placing imaging films on top of the membrane. Exposure is repeated, varying the time
as needed for optimal detection.
Crystal violet proliferation assay
Crystal violet (CV) is a triphenylmethane dye (4-[(4-dimethylaminophenyl)-phenyl-
methyl]-N,N-dimethyl-aniline) also known as Gentian violet (or hexamethyl
pararosaniline chloride). The crystal violet assay is useful for obtaining quantitative
information about the relative density of cells adhering to multi-well cluster dishes
thanks to its ability to bind to cells DNA.
For our experiments 2x104 cells have been plated in 24w multiwells and stimulated as
previously described. Then cells have been washed with PBS and incubate with crystal
violet solution for 5 minutes at 37 C°. The crystal violet solution contains 0,5% crystal
violet in deionized water and methanol 20%. After incubation crystal violet was
removed thorugh aspiration and three washings in PBS solution.
118
Finally, crystal violet uptaken by cells was solubilized after incubation in slow agitation
for 1 h at 37 C° with a solution containing sodium citrate 0,1 M, pH 4,2. After
incubation, solution containing solubilized crystal violet was collected and its
absorbance was evaluated at a 595 nm wavelenght. Each measurement was made in
triplicate for each point of the curve of growth. Absorbance is positively correlated to
crystal violet amount bound to cells DNA content.
Annexin V/Iodidium Propide cytofluorimetric staining
20x104 cells were plated in 60 mm Petri plates (p60) and treated with the different
nutrients as previously described.
Cells were then washed in PBS solution and detached from plates with Accutase
solution. The advantages of Accutase over the traditional Trypsin/EDTA treatment are
that it is less damaging to cells. In this case cells treatment with Accutase carries lower
risk to disrupt Annexin V antigens, expressed on the outer side of cell membranes
during apoptotic events.
After cells removal from the plastes, they have been respuspended in PBS solution and
centrifugated at 1000 rpm for 3 minutes. Finally, pelletted cells were resuspended in
100 µl containing 1 µl of annexin V, 1 µl of iodidium propide and 98 µl of buffer
solution, all provided by the kit. After 15 minutes of incubation at room temperature,
cells were evaluated by flow citometry for Annexin V/iodidium propide staining.
Intracellular ROS evaluation
For the evaluation of intracellular ROS amount, staining with 2',7'-
dichlorodihydrofluorescein diacetate (H2DCFDA) probe was performed. H2DCFDA is a
chloromethyl derivative of H2DCFDA. H2DCFDA passively diffuses into cells, where
its acetate groups are cleaved by intracellular esterases and its thiol-reactive
chloromethyl group reacts with intracellular glutathione and other thiols. H2DCF-DA is
solubilized in DMSO at moment of usage and avoided from light exposure.
For our experiments, 1,5x104 cells were plated on 6 cm plates and stimulated as
previously described, then stimulation culture medium was removed and fresh serum-
free DMEM was added. Then H2DCF-DA (5 g/ml) probe was added and incubated for
3 minutes at 37°C. After the incubation time, cells are blocked through PBS washing,
119
quickly lisated in RIPA lysis buffer supplemented with proteases inhibitors and kept for
1 minute at 4°C. Lysates were then collected and centrifugated for 1 minute at 13000
rpm. For each sample 100 µl were transferred on a 96 wells multiwell and analyzed and
fluorimetric quantification was performed at an excitation wavelength of 488 nm, while
the emission wavelegth read was set at 510 nm. Obtained values were finally
normalized on protein content of each sample.
Cell transfection with lipofectamine
Cells plated at a 90% confluence were transiently transfected using lipofectamine.
Before transfection, DMEM cultur medium is removed and replaced with Optimem
medium, lacking serum and antibiotics that could interfere with liposomes formation.
Acoording to Petri plates diameter (60 or 100 mm), 4 g or 12 g of siRNA are diluted
in 0.5 ml or 1.5 ml of Optimem medium and 10 g or 30 g of lipofectamine in 0.5 ml
or in 1.5 ml of Optimem medium. SiRNAs are used at a final concentration of 200 nM
for 10 µg di lipofectamine. After a 5 minutes incubation, solution containing siRNA is
added to solution with lipofectamine and incubated at room temperature for 20 minutes,
in order to promote liposomes formation, then equal amounts of final solution are added
to each plate. Optimem medium was removed after 4-6 h from transfection, as
lipofectamine could be slightly toxic for cells. Finally, cells were maintained in
complete medium for 48 h and transfection efficiency was evaluated through
immunoblotting assays using specific antibodies for the silenced protein.
Invasion assay
Transwell system, equipped with 8-μm pore polyvinylpirrolidone-free polycarbonate
filters, was used. Cells (2 × 104 in 200 μl) stimulated as previously described were
loaded into the upper compartment in serum-free growth medium, with or without 50
μmol/L ilomastat. The upper sides of the porous polycarbonate filters were coated with
50 μg/cm2 of reconstituted Matrigel basement membrane in order to mimic an ECM and
placed into six-well culture dishes containing 1 ml of complete growth medium. After
24 h of incubation at 37°C, noninvading cells and the Matrigel layer were mechanically
removed using cotton swabs, and the microporous membrane was fixed in 96%
methanol and stained with Diff-Quick solution. Chemotaxis was evaluated by counting
120
the cells that migrated to the lower surface of the polycarbonate filters (six randomly
chosen fields, mean ± SD).
Real-Time PCR
Total RNA was extracted from Hs29-4T and A375 derived from our experimental
conditions using the RNeasy Minikit kit. Total RNA (1 μg) was reverse-transcribed
using the Quantitect Reverse Transcription Kit. Reverse transcription was performed in
a final volume of 20 µl containing reverse transcriptase, real-time buffer 1x and real-
time primer mix. The amplification was carried out at 42°C for 2 minutes, then 42 C°
for 15 minutes and 95°C for 3 minutes. Measurement of gene expression was performed
by quantitative real-time PCR (7500 Fast Real-Time PCR System, Applied
Biosystems), using the Qiagen Quantifast SYBR Green PCR kit. For each sample, 1 µg
of cDNA was added to 25 µl of PCR mix. The samples were then subjected to 40 cycles
of amplification at 95°C for 10 s and 60°C for 30 s. RNeasy Minikit, Quantitect Reverse
Transcription Kit, all primer/probe mixes, and Qiagen Quantifast SYBR Green PCR Kit
were from Qiagen, except the following primers:
ADRB1 FW: 5’-CAGGTGAACTCGAAGCCCAC-3’
ADRB1 REV: 5’-CTCCCATCCCTTCCCAAACT-3’
ADRB2 FW: 5’-ACGCAGTGCGCTCACCTGCCAGACT-3’
ADRB2 REV: 5’-GCTCGAACTTGGCAATGGCTGTGA-3’
VEGF FW: 5’-TACCTCCACCATGCCAAGTG-3’
VEGF REV: 5’ ATGATTCTCCCTCCTCCTTC-3’
IL-8 FW: 5’-CTGGCCGTGGCTCTCTTG-3’
IL-8 REV: 5’-TTAGCACTCCTTGGCAAAACTG-3’
MMP-2 FW: 5’-ACGACCGCGACAAGAAGTTAT-3’
MMP-2 REV: 5’-ATTTGTTGCCCAGGAAAGTG-5’
MMP-9 FW: 5’-GACAAGCTCTTCGGCTTCTG-3’
121
MMP-9 REV: 5’-TCGCTGGTACAGGTCGAGTA-5’
IL-6 FW: 5’-AGTTCCTGCAGTCCAGCC-3’
IL-6 REV: 5’-TCAAACTGCATAGCCACTTTC-3’
Quantitative MMPs activity assay
Matrix metalloproteinases (MMPs) activity was measured with AmpliteTM
Universal
Fluorimetric MMP Activity Assay Kit according to the manifacturer’s instructions.
Briefly, serum-free medium from confluent monolayer of cells was collected and 5 µl
were added to 4-aminophenylmercuric acetate (AMPA; 1 nmol/L) at 37 °C for 1 hour to
detect MMP-2 activity and at 37 °C for 2 hours to detect MMP-9 activity. A 50 µl
portion of the mixture was then added to 50 µl of MMP Red substrate solution. After 60
minutes of incubation the signal was read by fluorescence microplate reader with
excitation (Ex)/ emission (Em) = 540 nm/590 nm.
Statistical Analysis
In vitro data are presented as means ± SD from at least three experiments. Results were
normalized versus control expression levels. Statistical analysis of the data were
performed by Student’s t test. p≤0.05 was considered statistically significant.
122
EXPERIMENTAL
PART I
123
AIM OF THE STUDY I
Stress is commonly defined as the general reaction of the organism in order to respond
to endogenous and exogenous stimuli that can affect the homeostasis of the organism
itself. The emotional arousal induced by the stimulus is processed in the CNS and
occurs both at a biological/somatic level, with autonomic and endocrine changes, and at
a psychological/behavioral level with sequences of motor behavior collectively known
as the "flight or fight response". This response is usually time-limited and is
progressively reduced when the stressful event is no longer present, but when stressors
keep sustaining secretion of stress mediators, we have a condition of chronic stress-
related disorder that constantly affects homeostatic mechanisms.
In the light of these observations, several epidemiological and clinical studies over the
years have demonstrated the close interrelationship between chronic stress, depression
and social isolation and cancer initiation and progression in several tumour models,
pointing out the role exerted by CA through adrenergic receptors activation in almost all
phases of cancer progression.
In particular, studies sight β-adrenergic receptor (AR) antagonists as novel therapeutic
agents for melanoma, as they may reduce disease progression. Here within, preliminary
studies have evaluated the expression of β-ARs in a series of human cutaneous
melanocytic lesions, and we therefore studied the effect of their endogenous agonists,
norepinephrine (NE) and epinephrine (E), on primary and metastatic human melanoma
cell lines. Using immunohistochemistry, it has been demonstrated that both β1- and β2-
ARs are expressed in tissues from benign melanocytic naevi, atypical naevi and
malignant melanomas and that expression was significantly higher in malignant
tumours.
Melanoma cell lines (human A375 primary melanoma cell line and human Hs29-4T
metastatic melanoma cell lines) also expressed β1 - and β2-ARs by measuring transcripts
and proteins. NE or E increased metalloprotease-dependent motility, released
interleukin-6 and 8 (IL-6, IL-8) and vascular endothelial growth factor (VEGF). These
effects of catecholamines were inhibited by the unselective β-AR antagonist
propranolol. The role of soluble factors elicited by catecholamines seemed pleiotropic
as VEGF synergized with NE increased melanoma invasiveness through 3D barriers,
while IL-6 participated in stromal fibroblast activation towards a myofibroblastic
phenotype. Our results indicate that NE and E produce in vitro via -ARs activation a
124
number of biological responses that may exert a pro-tumourigenic effect in melanoma
cell lines. The observation that -ARs are up-regulated in malignant melanoma tissues
support the hypothesis that circulating catecholamines NE and E, by activating their
receptors, favour melanoma progression in vivo.
125
RESULTS I
Preliminary results: expression of β-ARs in tissue sample
Melanoma represents the most aggressive type of skin cancer, with an increasing
incidence found especially in young adults. Surgery continues to provide a cure for
localized and regional disease. Despite increasing improvement in early diagnosis, a
significant reduction in mortality has not been observed yet, as metastatic melanoma is
still characterized by high resistance to drug therapies and radiation (MacKie et al.,
2009; Rughani et al., 2013). Therefore, at present there is no therapeuthic approach for
the complete cure of metastatic melanoma and the only effective treatment for the
eradication of the disease is represented by early phase surgery (Atallah et al., 2005).
Hence, in the last decade several studies have contributed to a better understanding of
the biological pathways underlying the process of melanoma dissemination and
metastasis in order to identify new therapeutic targets.
There is growing evidence that some stress neurotransmitters, such as norepinephrine
(NE) and epinephrine (E), directly contribute to promote tumour cell growth and
invasion, at least in part through -AR activation, thereby suggesting an important
neuro-oncological link in tumour progression (Sood et al., 2006; Yang et al., 2006;
Shang et al., 2009). This interrelationship among psychosocial stress, tumor growth and
β-adrenergic activation has also been confirmed in vivo in mice under controlled stress
conditions (Hasegawa and Saiki, 2002). Moreover, it has been shown that various
human solid tumours, such as breast, colon, prostatic, ovary, nasopharyngeal and oral
cancer, express β2-adrenoceptor (β2-AR), raising the possibility that such receptors may
affect proliferation, invasion and dissemination processes (Sood et al., 2006; Palm et al.,
2004; Drell et al., 2003). Interactions between tumour cells and soluble factors
originated from the nervous system has recently been proposed to favour metastasis
formation (Voss et al., 2010). Improved survival rates have been demonstrated in mice
with metastatic tumour by combined administration of -AR antagonists (Glasner et al.,
2010). In addition, recent evidence suggests a dramatic role of -AR blockers in
reducing metastases, tumour recurrence and specific mortality in breast cancer patients
(Powe et al., 2011). More recently, the use of β-blockers for concomitant disease was
associated with a reduced risk of progression of thick melanoma and with an increased
survival time of melanoma patients, suggesting that the interaction of catecholamines
with β-ARs could be a useful target in this disease (De Giorgi et al., 2011; Lemeshow et
126
al., 2011). However, no detailed information regarding -ARs expression in human
cutaneous benign and malignant melanocytic lesions or catecholamine influence on
melanoma cell migration has been provided so far.
According to these observation, studies conducted in collaboration between the Section
of Clinical, Preventive and Oncologic Dermatology and the Division of Pathological
Anatomy in the Department of Critical Care Medicine and Surgery of Florence by prof.
Silvia Moretti and prof. Daniela Massi evaluated the expression levels of both β1 and β2-
AR in order to point out a possible correlation between adrenergic receptors expression
and melanoma progression. To this aim, the study series included 5 common
melanocytic naevi (CN) (2 females, 3 males, age 28-54 yrs, mean 35.8 yrs); 5 atypical
(so-called dysplastic) melanocytic naevi (AN) (2 females, 3 males, age 30-47 yrs, mean
40.4 yrs); 5 in situ primary melanoma (PM) (2 females, 3 males, age 37-55 yrs, mean
44.2 yrs; site: 3 trunk, 1 lower extremity); 9 superficial spreading (SS) PM (7 females,
2 males, age 41-82 yrs, mean 58.4 yrs; site: 4 trunk, 3 leg, 2 arm; thickness 0.30-1.90
mm, mean 0.82 mm; 5 level II, 3 level III); 6 nodular (N) PM (3 females, 3 males, age
53-76 yrs, mean 61.5 yrs; site: 3 trunk, 1 leg, 2 arm; thickness 1.40-17 mm, mean 5.2
mm; 2 level III, 3 level IV, 1 level V), 10 metastatic melanoma (MM), 5 cutaneous and
5 lymph-nodal (1 female, 9 males, age 59-87 yrs, mean 77.1 yrs).
The presence of β-ARs was demonstrated in all melanocytic lesions examined. The
specimens were obtained by surgical resection in all cases and fixed in 10% formalin
before being processed in paraffin. Haematoxylin-eosin stained sections from each
histological specimen were reviewed to confirm the histological diagnosis. The
percentage of positive cells per lesion was scored according to semi-quantitative
criteria. Since the percentage of positive naevus melanocytes/melanoma cells was
always higher than 50%, semi-quantitative results were expressed as score 1 (50-80%
positive naevus melanocytes/ melanoma cells), score 2 (81-90% naevus
melanocytes/melanoma cell staining ), and score 3 (91-100% melanoma cell staining).
The cell staining intensity was scored on a scale as weak, moderate, strong, very strong.
In particular, the staining for 1-AR was confined to the cell cytoplasm in naevus
melanocytes and melanoma cells; the reaction for 2-AR was also confined to the cell
cytoplasm in all cases, with an additional peripheral membrane pattern in some AN
melanocytes and malignant cells. The cell staining intensity was always weak with
regard to the reaction for β1-AR, whereas the immunostaining for β2-AR appeared to be
weak in CN, moderate (except one very strong reaction), in AN; moderate or strong in
127
in situ PM; from weak to moderate or strong in SSPM, and from strong to very strong in
NPM and MM, with no difference between cutaneous and nodal metastasis.
In regards to β1-AR expression, score 1 was evaluated in both CN and AN, score 2 was
found in a minority (3 in situ and 2 SS) of PM, whereas in the other PM and MM score
3 was detected. β1-AR expression was significantly higher in malignant than in benign
lesions (p0.0001) and in PM or MM than in naevi (p0.0001 and p0.0001). No
difference was observed between CN and AN, or between in situ/SSPM compared to
NPM/MM.
With regards to β2-AR expression, score 1 was observed in CN, score 2 in AN and score
3 was detected in all PM and MM but one (SSPM), which exhibited score 2. β2-AR
reactivity was significantly higher in malignant lesions than in naevi (p0.0001), and in
PM or MM respectively, than in naevi (p0.0001 and p0.0001). AN exhibited a
significantly higher reactivity compared to CN (p0.003), and no difference was
observed between in situ/SSPM and NPM/MM. In addition, no significant difference
was detected between PM and MM for both receptors.
Epidermal keratinocytes were lightly coloured for β2-AR. In fact, it has been previously
described that human keratinocytes primarily express β2-AR (Sivamani et al., 2007).
Endothelial and stromal cells exhibited heavy reactivity for 2-AR in malignant lesions,
and to some extent, in AN.
Taken together, these preliminary data showed that β1- and β2-ARs were variably
expressed in human melanocytic lesions with a significant up-regulation in PM and
MM, and, at least for β2-AR, a significant up-regulation was also observed in AN versus
CN.
128
β-AR expression analysis and responsiveness of melanoma cell lines after
catecholamines stimulation
In order to confirm the correlation between sensitivity to catecholamines and
progression towards a malignant phenotype of melanoma cells hypothesized after the in
vivo preliminary analysis, we performed our in vitro experiments on two different
melanoma cell lines, namely Hs29-4T cells, selected from a lymph nodal metastatic
lesion, and A375 cells, derived from human primary melanoma.
First we evaluated expression levels of both β1 and β2-AR on both cell lines, performing
Western Blot analysis and evaluation of mRNAs through Real Time PCR analysis. We
observed that both cell lines express low and comparable levels of β1-AR, as shown in
Figure 1, while they both express higher amounts of β2-AR (Figure 2A and 2B), with
the primary A375 melanoma cell line exhibiting a significantly higher expression of β2-
AR compared to the metastatic Hs29-4T cell line.
Fig. 1. Expression of β1-AR in melanoma cell lines. (A) Analysis of β1-AR expression by immunoblot in
primary (A375) and metastatic (Hs29-4T) melanoma cell lines. (B) Amount of ADRB1 mRNA by real-
time PCR. The amount of target, normalized to the endogenous reference (18S RNA), was given by the 2-
ΔΔct calculation and was reported as 2
-ΔΔct. Both immunoblots and real-time PCR are the mean of three
independent assays.
A375 Hs29-4T
- 72
- 55
- 36
A B
129
Fig. 2. Expression of β2-AR in melanoma cell lines. (A) Analysis of β2-AR expression by immunoblot in
primary (A375) and metastatic (Hs29-4T) melanoma cell lines. (B) Amount of β2-AR mRNA by real-
time PCR. The amount of target was given by the 2-ΔΔct
calculation and was reported as 2-ΔΔct
.
Immunoblot and Real-Time PCR are the mean of three different experiments.
The second messenger cyclic AMP (cAMP) mediates several cellular responses to
external signals such as proliferation, ion transport, regulation of metabolism and gene
transcription by activation of the cAMP-dependent protein kinase (cAPK or PKA).
Activation of PKA occurs when two molecules of cAMP binds to the two regulatory
subunits of the tetrameric PKA holoenzyme, leading to release of active catalytic
subunits. Activation of transcription upon elevation of cAMP levels results from
translocation of PKA to the nucleus where it phosphorylates serine or threonine residues
on target proteins that bear PKA responsive motif R-R-X-(S/T)-Y like the transcription
factor CREB, which in turn leads to TFIIB binding to TATA-box-binding protein
TBP1, thus linking phosphorilated CREB to the Pol II transcription initiation complex
(Taylor et al., 1990).
As already known, NE secretion leads β adrenergic receptors stimulation, resulting in
Gαs-mediated activation of adenylyl cyclase and subsequent conversion of ATP into
cAMP. Transient flux of intracellular cAMP activates PKA to phosphorylate multiple
target proteins, including transcription factors of the CREB/ATF and GATA families, as
well as β adrenergic receptor kinase (BARK). BARK recruitment of β-arrestin inhibits β
adrenergic receptor signaling and activates Src kinase, resulting in activation of
transcription factors such as STAT3 and metastasis-associated genes involved in
Hs29-4T A3750
1
2
3
4
5
2 -
C
T
Hs29-4T A375
A B
130
inflammation, angiogenesis, tissue invasion, and epithelial–mesenchymal transition
(EMT), and in downregulation of genes promoting antitumour immune responses (Cole
and Sood, 2012).
According to these observation, we performed a Western Blot analysis of activated
PKA level in order to assess melanoma cell lines responsiveness to NE stimulation. The
doses of NE and E used for our study were selected to reflect the physiological
conditions of this hormone at the level of the tumour. Although circulating plasma
levels of NE are only 10 to 1000 pM in a normal individual, they may reach as high as
100 nM in conditions of stress (Schmidt et al., 1996). Studies suggest that within the
tumour microenvironment, concentrations may reach as high as 10μM (Lara et al.,
2002). In our case, pilot experiment show best responsiveness of both cell lines at a
catecholamines concentration of 1 µM. As shown in figure 3A and 3B, both cell lines
are able to respond to catecholamine stimulation with PKA activation in a range of time
between 5 and 20 minutes of stimulation, while at 30 minutes the signal starts
decreasing, according to a progressive receptor signaling downregulation.
Fig. 4 (A, B). Activation of PKA. Melanoma cell lines were serum-deprived overnight and then
stimulated with NE (1 µM) for the indicated period and an immunoblot analysis for the detection of the
phosphorylation level of p-PKA was shown. Actin immunoblot was used for normalization. The bar
graph below represents the phosphorylation level of PKA in four different experiments. *p<0.005.
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
0,45
c 5' 10' 20' 30'
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
0,45
c 5' 10' 20' 30'
Hs29-4T A375
A375 Hs29-4T
ctrl 5 10 20 30 ctrl 5 10 20 30
Actin
min NE
α-p-PKA
C
D
131
To further elucidate the role of catecholamines in melanoma signaling, we evaluated the
actiovation of the mitogen-activated protein kinase (MAPK) pathways. Several studies
have in fact related β adrenergic-dependent MAPK signaling to increased growth and
aggressiveness of breast (Cakir et al., 2002), pulmonary (Schuller and Cekanova, 2005)
and pancreatic carcinoma (Weddle et al., 2001; Askari et al., 2005), but few studies
until now have investigated the effects of β-adrenergic signaling on these molecular
pathways in melanoma models. Thus we performed a Western Blot analysis in order to
evaluate phosphorylated levels of both p42-p44 and p38 MAPK. In particular, we
analysed phosphorilation of Thr202 and Tyr404, key aminoacidic residues for MAPK
activation.
Our data demonstrate that A375 cells show a progressive activation of p42/p44 MAPK
that reaches its maximal level at 30 minutes of stimulation with both E and NE (figure
4A and 4B). On the other hand, Hs29-4T cells respond to E stimulation within 15
minutes of stimulation, while after 20 minutes p42/p44 phosphorilated levels start
decreasing. On the contrary, they show a similar activation pattern to A375 cells after
NE stimulation, where p42/p44 reach maximal activation after 30 minutes (figure 4A
and 4B).
For what concerns p38, also in this case both NE and E are able to elicit its activation in
both cell lines, although with different kinetics (fig. 4A and 4B).
c 5' 10' 15' 20' 30' c 5' 10' 15' 20' 30'
0,0
0,2
0,4
0,6
0,8
1,0
1,2
A375 Hs29-4T
p-p
42p
44 /
p42p
44 r
ati
o
Minutes of stimulation with E
c 5' 10' 15' 20' 30' c 5' 10' 15' 20' 30'0,0
0,2
0,4
0,6
0,8
A375 Hs29-4T
p-p
38 /
p38
rati
o
Minutes of stimulation with E
A A375 Hs29-4T
ctrl 5 10 15 20 30 ctrl 5 10 15 20 30
p-p42/p44 MAPK
p42/p44 MAPK
minutes of stimulation with E
p-p38
p38
132
Figure 4. Analysis of activation of p42/p44 and p38 MAPK. Melanoma cell lines were serum-deprived
overnight and then stimulated with E (A) or NE (1 µM) (B) for the indicated period and an immunoblot
analysis for the detection of the phosphorylation level of MAPKs were shown. Total p42/p44 and p38
MAPK immunoblot were used for normalization. The bar graphs below represent the phosphorylation
level of MAPKs in four different experiments. *p<0.005.
Taken together, our data further confirm that both primary and metastatic cell lines
respond to catecholamines promoting activation of both PKA and p42/p44 and p38
MAPK pathway, acknowledged to play mandatory roles in regulating cancer cells
growth, survival and invasive ability.
B
A375 Hs29-4T
ctrl 5 10 15 20 30 ctrl 5 15 20 30
p-p42/p44 MAPK
p42/p44 MAPK
minutes of stimulation with NE
p-p38
p38
10
c 5' 10' 15' 20' 30' c 5' 10' 15' 20' 30'0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
A375 Hs29-4T
p-p
42p
44 /
p42p
44 r
ati
o
Minutes of stimulation with NE
c 5' 10' 15' 20' 30' c 5' 10' 15' 20' 30'0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
A375 Hs29-4T
p-p
38 /
p38 r
ati
o
Minutes of stimulation with NE
133
Catecholamines increase melanoma cells invasive ability
Activation of autocrine, paracrine and/or endocrine pathways At both primary and
metastatic tumour sites can promote tumour cell proliferation by disrupting the balance
between positive, pro-proliferative and pro-invasive, and inhibitory signals (Langley
and Fidler, 2007). As already stated, several studies have demonstrated the close
interrelationship between psychosocial factors and cancer progression. In particular,
chronic stress and related continuative production of stress hormones are able to affect
key tumour cells mechanisms like invasive/metastatic ability and angiogenesis in order
to promote tumour spreading in distant sites. For example, it has been demonstrated in
models of ovarian and nasopharyngeal carcinomas that NE increases cells motility and
invasive ability through upregulation of MMPs secretion (Yang et al., 2006; Sood et al.,
2006). In addition, the β blocker propanolol showed to have effect in impairing cancer
cells invasion through inhibition of MMPs and VEGF secretion in an in vitro model of
pancreatic carcinoma (Guo et al., 2009).
According to these observation, we therefore analysed the motility of melanoma cells
upon catecholamine stimulation. The invasion assay was carried out by Boyden
chambers placed in a well of a 24 multiwell. Cells were seeded on the upper part of the
filter, while a chemoattractant stimulus, in this case culture medium supplemented with
serum, was placed in the bottom of the well. In order to mimick the basal layer
structure, the Boyden filter was covered with Matrigel, syntethic mixture of different
structural proteins like laminin, entactin, proteoglycans and collagen, thereby creating a
three-dimensional layer. After 24 h of stimulation with catecholamines, filters were
subjected to haematoxylin-eosin staining in order to evaluate the number of cells
migrated to the lower side of the Boyden filter. We selected adequate time point by
means of pilot experiments showing 24 h as the best to obtain motility increase.
As showed in bar graphs in figure 5A and 5B, both NE and E are able to elicit invasive
behaviour in both metastatic and primary melanoma cells. In particular, both NE and E
appear to be more effective in primary melanoma cells with respect to metastatic
melanoma cells, thereby according to an increase in cancer aggressive behaviour. In
addition, E is the most efficient catecholamine in eliciting invasiveness of the metastatic
Hs29-4T cell line. In order to further confirm the involvement of β subtypes of
adrenergic receptors activation in modulating melanoma cells invasion, A375 and Hs29-
4T cells were pre-treated with β blocker propanolol before performing stimulation with
catecholamines, and seeded in the upper side of the Boyden chamber. As shown in
134
figure 5A and 5B, propanolol impairs invasive ability of both primary and metastatic
melanoma cells, thereby reducing the number of invaded cells to control levels.
Given their ability to degrade the ECM, MMPs act directly on the motility and
invasiveness of cancer cells, allowing them to overcome the boundaries of the tissues
and therefore to exit from the tumour primary site and reach blood and lymphatic
vessels in order to metastatize in distant tissues (Sternlicht et al. 1999; Boire et al.
2005). To further confirm the role of MMPs in mediationg catecholamines-dependent
cells motility, we performed Boyden assays incubating both cells lines with ilomastat, a
broad range pharmacological inhibitor of MMPs activity. As demonstrated by both
pictures and bar graphs in figure 5A and 5B, the pro-invasive effect of both NE and E is
strongly sensitive to ilomastat, that reduces the number of invaded cells to control
levels.
untre
ated N
E
NE +
ILO
NE +
Pro
p --
untre
ated N
E
NE +
ILO
NE +
Pro
p
0
200
400
600
800
1000
1200
1400
A375
Hs29-4T
n.
cells
mig
rate
d t
hro
ugh m
atr
igel
Untreated
NE 1 µM
NE + Ilomastat
50 µM/L
NE + Propranolol
1 µM
Hs29-4T A375
Figure 5A. Melanoma cell lines were
serum-deprived overnight and then seeded
in the upper Boyden chamber for assay
their invasion. NE (1 µM), in presence or
absence of ilomastat (50 μmol/L) or
propranolol (1µM), was added in the upper
Boyden chamber. Bar graphs represent the
mean of migrated cells counted in six
different fields for each experiment.
*p<0.005 versus untreated.
135
Taken together, our data demonstrate that catecholamines stimulation promotes
melanoma cells invasion through activation of a β-adrenergic-dependent pathway that
involves MMPs secretion, thereby confirming the involvement of a proteolytic
degradation of the matrigel barrier during invasion.
We therefore analysed by Real Time PCR the expression of MMP-2 and MMP-9, the
main proteolytc enzymes expressed by Hs29-4T and A375 cell lines. Melanoma cell
untre
ated E
E + IL
O
E + P
rop --
untre
ated E
E + IL
O
E + P
rop
0
200
400
600
800
1000
A375
Hs29-4T
n c
ells
mig
rate
d t
hro
ugh m
atr
igel
Untreated
E 1 µM
E + Ilomastat 50
µM/L
E + Propranolol 1
µM
Hs29-4T A375
Figure 5B. Melanoma cell lines were
serum-deprived overnight and then
seeded in the upper Boyden chamber
for assay their invasion. E (1 µM), in
presence or absence of ilomastat (50
μmol/L) or propranolol (1µM), were
added in the upper Boyden chamber.
Bar graphs represent the mean of
migrated cells counted in six different
fields for each experiment. *p<0.005
versus untreated.
136
lines were serum-deprived overnight and then stimulated with E or NE 1µM for 24 h.
At the end of the stimulation, RNA was extracted and analysed in order to maintain the
same analysis time point as the one used for motility assays. Figure 5C and 5D reveal
that, while NE and E do not influence MMP-9 production, the expression of MMP-2 is
increased by NE in A375 primary melanoma cells and by E in metastatic Hs29-4T.
Figure 5C and 5D. Expression of MMP-2 (C) and MMP-9 (D) mRNA by real-time PCR. Melanoma cell
lines were serum-deprived overnight and then stimulated with E or NE (1µM) for 24 h. The amount of
target, normalized to the GAPDH mRNA amounts, was given by the 2-ΔΔ
CT calculation and was reported
as arbitrary units (a.u.). The graphs reports data as the mean of three independent assays.
In order to further elucidate this data apparently in contrast with several studies in
literature, we analysed enzymatic activity of MMP-2 and MMP-9 with a fluorimetric
kit. This MMP activity assay kit uses a Tide Fluor™ 3 (TF3)/Tide Quencher™ 3 (TQ3)
fluorescence resonance energy transfer (FRET) peptide as a MMP substrate. In the
intact FRET peptide, the fluorescence of TF3 is quenched by TQ3. Upon cleavage into
two separate fragments by activated MMPs, the fluorescence of TF3 is recovered and its
signal can be easily read by a fluorescence microplate reader at Ex/Em = 540/590 nm.
Our data demonstrate that both NE and E are able to maintain a high activation state of
secreted MMP-2 and MMP-9 following catecholamines stimulation for 24h on both cell
lines (figure 5E and 5F).
untreated NE E -- untreated NE E
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
A375Hs29-4T
MM
P-2
exp
ressio
n
untreated NE E -- untreated NE E
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
A375Hs29-4T
MM
P-9
expre
ssio
n
137
Figure 5E and 5F. MMP-2 and MMP-9 activity assay. Melanoma cell lines were serum-deprived
overnight and then stimulated with NE (A) or E (1 µM) (B) for 24h. The media obtained were then tested
for MMPs activity with a fluorimetric kit, following the manifacturer’s instructions. Data are presented as
RFU versus concentration of test compounds. The graphs reports data as the mean of four independent
experiments. *p<0.005.
In conclusion, data obtained so far give new insights about the role of catecholamines in
influencing MMPs activity both at a transcriptional and post-translational level. Indeed,
we observed a prolonged and sustained activation of both MMP-2 and MMP-9 by
catecholamines in melanoma cells, while analysis of RNA revealed a control of
expression, mainly focused on MMP-2.
untreated NE E -- untreated NE E
0,0
0,5
1,0
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3,0
A375Hs29-4T
RF
U (
Ex5
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MMP-2 activity assay
untreated NE E -- untreated NE E
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138
Effects of catecholamines on cytokine production and tumour microenvironment
Cytokines are key molecules secreted by the immune system and play important roles in
the cross-talk between different immunologically active cells and tissues, but cytokines
are secreted also by non immune-related cells like endothelial and epithelial cells, thus
mediating systemic responses to physiological or pathological stimuli.
In the light of these observation, it has been demonstrated that cancer cells secrete many
cytokines, chemokines and growth factors that can affect their own aggressiveness, in
terms of proliferation, invasion or survival, as well the reactivity of the surrounding
stroma. For example, cancer cells production of IL-8 promotes proliferation and
survival of cancer cells through autocrine signaling pathways. In addition, tumour-
derived IL-8 activates endothelial cells in the tumour vasculature to promote
angiogenesis and induce a chemotactic infiltration of neutrophils into the tumour site
(Wilson and Waugh, 2008). Also IL-6 plays an important role both in regulating
proliferation, invasiveness/scattering and resistance to apoptosis of tumor cells. IL-6 has
also been shown to enhance endothelial cell migration, a key step in angiogenesis, and
dissemination of solid tumors, also through a synergistic action with VEGF, main
promoter of cancer angiogenic sprouting. Thus increased expression of these cytokines,
either in the circulation or in tumour tissue, is correlated with worse prognosis in several
cancers (Dankbar et al., 2000; Ara et al., 2010; Otrock et al., 2011).
To address the role of catecholamine stimulation in these features we first analysed the
expression of a panel of cytokines/growth factors by quantifying mRNA levels thorugh
real time PCR analysis after stimulating cells for 24 hours with NE or E. We found that
catecholamine stimulation leads to an increase in the expression of VEGF, IL-6 and IL-
8 in both cell lines (Figure 6A, 6B, 6C). Interestingly, the two catecholamines used
show differential effects for VEGF, IL-6 and IL-8 in Hs29-4T and A375 cells. As
shown in the bar graphs, A375 primary melanoma cells respond to both NE or E
eliciting an almost twenty-fold higher expression of IL-6 and an almost five-fold greater
expression of VEGF and IL-8. Conversely, the Hs29-4T metastatic cell line senses NE
to increase expression of IL-6 and IL-8, while E seems to play a more important role in
promoting VEGF expression.
139
Figure 6. Expression of VEGF, IL-6 and IL-8 in E- and NE-treated melanoma cell lines. Melanoma cell
lines were serum-deprived overnight and then stimulated with E or NE (1µM). Total RNA was used for
the amplification of mRNA of IL-6 (A), VEGF (B) and IL-8 (C), using as housekeeping gene GAPDH
mRNA. The amount of target, normalized to the GAPDH mRNA amounts, was given by the 2-ΔΔCT
calculation and was reported as arbitrary units (a.u.). The graphs reports data as mean of three
independent assays. *p<0.005.
As already stated, cancer cells keep creating a complex and continuative “cross-talk”
with surrounding, non-malignant cells and/or with the extracellular architecture through
direct cell-to-cell contacts and paracrine/exocrine signals. These interactions are not
static, but they evolve along with tumour progression. For example, CAFs express
various growth factors and cytokines like IGF-1 and HGF that promote survival,
B
C
untreated NE E -- untreated NE E
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IL-6
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140
migration and invasion of tumour cells (Li et al. 2003; De Wever et al. 2004; Lewis et
al. 2004).
In order to evaluate a possible synergy between catecholamines stimulation and
cytokines in promoting primary melanoma aggressiveness, we performed invasion
assays where A375 primary melanoma cells were seeded in the upper Boyden chamber
and stimulated with NE (1 µM) alone or in combination with IL-6 (50 ng/µl) or VEGF
(20 ng/µl). We observed that, while VEGF and IL-6 alone do not affect cancer cells
invasion, stimulation with VEGF increases the invasive spur induced by NE in A375
primary melanoma cells. On the contrary, combined A375 cells stimulation with NE
and IL-6 does not seem to play a significant role (Figure 7A).
Figure 7A. Synergy among NE, cytokines and tumour microenvironment. (A) A375 primary melanoma
cells were serum-deprived overnight and then seeded in the upper Boyden chamber for assaying their
invasion. NE (1 µM), IL-6 (50 ng/µl), VEGF (20 ng/µl), in combination with NE or alone, were added in
the upper Boyden chamber. Bar graph represents the mean of migrated cells counted in six different fields
for each experiment. *p<0.005 versus untreated.
A
untreate
dNE
VEGF
IL6
NE + V
EGF
NE + IL
-6
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igra
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rigel
141
As a result of specific environmental stimuli produced by neighboring cancer cells,
healthy fibroblasts may undergo an activated state, named “myofibroblastic”, which is
characterized by the de novo expression of α-smooth muscle actin (α-SMA) protein, the
actin isoform typical of smooth muscle cells. Among molecules produced by tumour
cells, IL-6 has been described as a key molecule in activating surrounding stromal
and/or inflammatory cells infiltrating the tumour burden (Ara et al., 2010).
We therefore analyzed the ability of IL-6, in association with NE, to induce an activated
state in healthy dermal fibroblasts. To this aim we performed a Western Blot analysis in
order to evaluate the protein level of α-SMA in human dermal fibroblasts (HDFs)
treated with conditioned media (CM) A375 cells. To obtain CM from melanoma cells,
A375 cells were stimulated in starvation medium with NE, IL-6 or VEGF. After 24
hours of stimulation, the media was removed and replaced with fresh serum-free culture
medium in order to obtain CM free from NE, IL-6 or VEGF, but conditioned by their
previous administration. HDFs were then incubated with the obtained CM for 24 h and
then used for Western blot analysis. We observed that IL-6 is able to activate dermal
fibroblasts, as demonstrated by their ability to elicit α-SMA expression in comparison
with the untreated condition. In addition, in dermal fibroblasts, the conditioned medium
of NE-treated A375 cells induce an activation state very similar with respect to
treatment with IL-6 alone. Conversely, VEGF treatment is almost ineffective in eliciting
a reactivity of fibroblasts (Figure 7B).
Figure 7B. Analysis of human dermal fibroblasts (HDFs) activation state through evaluation of α-SMA
expression. A375 cells were grown to sub-confluence and treated for 24 h with 1 µM NE, IL-6 (50 ng/µl)
or VEGF (20 ng/µl). Fresh serum-free medium was added for additional 24 h before collection of
conditioned medium (CM) free from NE, IL-6 or VEGF, but conditioned by their earlier administration.
HDFs were then incubated with the obtained CM for 24 h and then used for Western blot analysis of α-
SMA expression. The blot is representative of three different experiments.
B
WB: α-SMA
Untr
eate
d
CM
+ V
EG
F
CM
+ I
L-6
CM
+ N
E
WB: Actin
142
Taken together, these data suggest that in vitro the treatment of human melanoma cells
with catecholamines dramatically affects their aggressiveness, inducing expression of
MMP-2, VEGF, IL-6 and IL-8. These factors orchestrate a feed-forward loop leading to
increase of proteolytic invasiveness of tumour cells, as well as activating surrounding
fibroblasts.
143
DISCUSSION I
The mediators of stress norepinephrine (NE) and epinephrine (E) exert stimulant effects
on the invasion process of a number of neoplastic cells, and this effect seems to be due,
at least in part, to the interaction with β-adrenoceptors (AR), which are expressed by
various malignant tumours. In particular, the expression of β-AR has been studied in
various human solid tumours, such as breast, colon, prostatic, ovary, nasopharyngeal
and oral cancer, raising the possibility that such receptors could affect invasion and
dissemination processes.
My study therefore aimed to evaluate the expression of β-AR and the influence of NE
and E on the malignant behaviour of melanoma cell lines. Preliminary results assessed
by immunohistochemistry on a series of human benign and malignant melanocytic
lesions showed that both β1 and β2-AR were expressed in melanocytic tumours,
according to the fact that both benign and malignant lesions can theoretically be
affected by catecholamines in vivo. Anyway, both β1 and β2-AR showed significantly
higher expression in malignant than benign lesions, and, for what β2-AR is concerned,
its expression is primarily related to atypical than common naevi. These findings
suggest that malignant lesions can be more deeply influenced by catecholamines than
benign counterparts, as the staining intensity for β2-AR progressively increased from
CN toward AN and to PM and MM, whereas the reaction intensity for β1-AR was
weaker in all groups of lesions. Such a difference can rely on a lower expression of β1-
AR than β2-AR on naevus melanocytes and melanoma cells. This hypothesis was
thereby supported by in vitro data, as both real time PCR and western blot analysis of
primary and metastatic melanoma cell lines demonstrated a lower expression of β1-AR
versus β2-AR.
We also demonstrated that A375 primary and Hs29-4T metastatic melanoma cell lines
respond to catecholamine stimulation enhancing motility and invasion and producing
molecules closely related to neoplastic progression. In keeping, we show that NE and E
are able to elicit activation of p42/p44 and p38 MAPKs, acknowledged to play
mandatory roles for cell growth, survival and invasive ability, in both primary and
metastatic cell lines. These data could be of striking interest in order to find new
strategies for melanoma treatment. In fact, Meier et al. showed that combined targeting
of the p42/p44 and Akt signaling pathways significantly inhibited growth and enhanced
apoptosis in melanoma cell cultures (Meier et al., 2007).
144
Moreover, the two cell lines tested show differential responsiveness to NE and E.
Concerning the invasion assay, the primary cell line responded to NE and, to a lesser
degree, to E, while the metastatic cell line showed a clear reaction only to E. The
inhibition induced by ilomastat strongly suggests a proteolytic degradation of matrigel
and the probable intervention of MMPs in both cell line invasive process. In fact, real
time PCR analysis demonstrates that MMP-2, rather than MMP-9, is produced at a
significantly higher level compared to baseline, by A375 cells under NE stimulus and
by Hs29-4T cells under E stimulation. Furthermore, our data show that both NE and E
are able to elicit sustained activation of MMP-2 and MMP-9 both in primary and
metastatic cell lines. Thus, increased motility of melanoma cells seems to be due to a
proteolytic invasive capacity, typical of a mesenchymal phenotype (Hoffmann et al.,
2000; Parri et al., 2009), and catecholamines seem able to influence MMPs activity both
at a transcriptional and at a post-translational level. This disparity could be explained by
the fact that activation of pro-MMP-2 and pro-MMP-9 is closely related, as it has been
demonstrated that active MMP-2 can convert pro-MMP-9 to its active form through
direct proteolytic action. In the light of these findings, MMP-9 activation could be a
downstream effect of MMP-2 direct regulation by catecholamines (Bauvois, 2012). The
migration of neoplastic cells appears to be increased through activation of β-ARs,
because it is completely abolished by β-ARs antagonist propanolol. As stimulation of
both cell lines with NE and E induces activation of the protein kinase PKA, we can
speculate that catecholamines exert their functions on melanoma cell lines primarily
through a PKA-dependent pathway.
Also for what the production of cytokines is concerned, NE and E show differential
effects on melanoma cell lines. In fact, primary A375 cells significantly increase levels
of IL-6 and VEGF under NE and E challenge, whereas metastatic Hs29-4T cells
increase IL-6 expression under NE stimulus, and produce significant amounts of VEGF,
especially under E activation. Concerning the expression of IL-8, the primary cell line
responded to both NE and E at a significantly higher degree compared to the metastatic
one, and both cell lines reacted more intensely to NE. This result is in agreement with
the angiogenic role of IL-8, since de novo angiogenesis is particularly useful for primary
tumours to escape the hostile microenvironment and disseminate metastasis (Singh and
Varney, 2000). In addition, the catecholamine-induced IL-8 enhancement is in
agreement with the described IL-8 stimulation produced by NE in prostate cancer (Voss
et al., 2010).
145
We do not know exactly why such a discrepancy exists between primary and metastatic
cell line response, but it is possible that at least in part this difference is due to a higher
expression of β2-ARs, assessed as protein and transcript, on the primary melanoma cell
line. Another possibility is that IL-6 and IL-8, whose expression was associated with
early malignancy of melanoma in vivo are actually secreted more efficiently by a cell
line derived from a primary melanoma, thereby leading to enhanced aggressiveness
towards a more metastatic phenotype (Moretti et al., 1999).
In vitro experiments clearly show that catecholamines can increase the malignant
behaviour of melanoma cells affecting both invasion capacity and cytokine production.
Preliminary data in melanoma tissue sections also showed a strong reactivity for β2-AR
in most endothelial and stromal cells, including macrophages, suggesting the possible
influence of catecholamines on cells of the tumour microenvironment. In keeping,
recent studies showed that phagocytic cells like neutrophils and macrophages are direct
sources of catecholamines and that stimulation with both NE and E can enhance
macrophagic release of proinflammatory cytokines as TNF-α, IL-1β and IL-6 through a
NF-kB-dependent pathway (Flierl et al., 2009). These data could support the hypothesis
of potential feed-forward biologic loops capable of affecting metastatic behaviour of
neoplastic cells.
Our in vitro experiments could further confirm that some pro-metastatic loops could
work in in vivo melanoma model too. In fact, we demonstrate that IL-6 and NE in
melanoma cells can activate dermal fibroblasts toward a myofibroblastic phenotype, as
demonstrated by upregulated -SMA expression (Kalluri et al., 2006). It is well known
that stromal fibroblasts within tumours undergo a process, commonly called
mesenchymal-mesenchymal transition (MMT) to myofibroblasts, leading them to a
more contractile phenotype and allowing a cross talk with tumour cells dramatically
affecting their aggressiveness (De Wever and Mareel, 2003; Dvorak et al., 2011). In
turn, activated fibroblasts can secrete other pro-metastatic cytokines, such as VEGF,
capable of inducing further tumour angiogenesis. In keeping with these observations, in
our experiments, VEGF can increase melanoma cell migration and invasion ability,
particularly when associated with NE (Dong et al., 2004).
Previous data support the hypothesis that various types of stress, such as surgical
procedure or neuroendocrine stress due to psycho-social factors, can stimulate tumour
progression both in animals and humans (Voss et al., 2010). This seems to be true also
for melanoma at least in in vivo models (Azpiroz et al., 2008; Vegas et al., 2006).
146
Our work provides evidence that stress hormones like NE and E can significantly
stimulate the malignancy of melanoma cells at various levels and that β-ARs, likely
involved in this response, are largely expressed in melanoma cell lines and cutaneous
melanomas. It is the first time, to our knowledge, that β-ARs are demonstrated in a large
series of human cutaneous melanocytic lesions, and even in melanocytic naevi,
suggesting a potential influence of catecholamines also in benign counterparts.
Moreover, the detection of β2-AR also in the stromal cells of melanoma
microenvironment implies further possible effects of catecholamines on melanoma
progression. Consequently, it is possible that the interaction catecholamines-β-ARs
could play a dramatic role during the clinical course of melanoma patients. The efforts
to understand molecular events underlying such an interaction could therefore be very
useful for indicating new targets in therapy.
147
CONCLUDING REMARKS I
On the basis of the results obtained we can speculate that in aggressive melanoma, the
action exerted by catecholamines both on the tumour cells and their microenvironment
could be related at least in part to the preservation of a tumour stem niche, which may in
turn contribute to resistance to conventional pharmacological therapies. At this purpose,
it has been recently reported that melanoma stem cells isolated from patients showed
higher expression of multiple ABC transporters and IL-8, suggested to be related with
increased chemoresistance of melanoma cells (Luo et al., 2012). Also VEGF directly
acts on the cancer stem niche through blood vessel formation via its primary function on
endothelial cells, and thus increasing the area of the niche required for preservation of
stem cell-like properties. In addition, VEGF can act on melanoma stem cells in an
autocrine way, promoting their self-renewal, through its receptor VEGFR-2 (Takakura,
2012). We can therefore hypothesize that catecholamines could promote melanoma
aggressiveness both enhancing angiogenesis and motile ability of cancer cells and also
providing a possible protective mechanism for melanoma stem cells from drugs
delivery, likely in an IL-8 and VEGF-dependent way. This fascinating hypothesis could
be strengthened by our recent observation that melanoma cells treated with NE and β3-
AR selective antagonist SR59230A show diminished percentage of cells positive to
stem marker CD133, thereby suggesting a role of β3-AR in controlling stem-like
properties of a small population of melanoma cells (Calvani M., unpublished data). In
the light of this hypothesis, also the action exerted by catecholamines on the tumour
microenvironment could be related to protection and prevention of premature
exhaustion of the stem cell niche with cancer cells characterized by stem cell-like
properties. To this purpose, it has been reported that myofibroblasts and endothelial
cells are key components of the tumour stem niche and support in vivo tumour initiation
from a small number of cancer stem cells (Vermeulen et al., 2010; Calabrese et al.,
2007). Our experiments showed that IL-6 and NA in melanoma cells can activate
dermal fibroblasts toward a myofibroblast phenotype, allowing a cross-talk with tumour
cells, thereby dramatically increasing their aggressiveness. In turn, activated fibroblasts
can secrete other pro-metastatic cytokines, such as VEGF and SDF-1, capable of further
inducing tumour angiogenesis and recruitment of distant cells, thus activating further
pro-metastatic circuits (De Wever 2003; Dvorak 2011; Dong 2004). This could be
confirmed in our model by recent observations that melanoma cells stimulated with NE
148
recruit CAFs, macrophages, adult endothelial cells and endothelial precursor cells
(EPCs) (Calvani M., unpublished data). Of note, both macrophages and endothelial cells
have been recently described as sources of catecholamines (Flierl et al., 2009; Sorriento
et al., 2012). Hence, both systemic and tumour-neighbouring sources of catecholamines
could promote recruitment and differentiation of CAFs and EPCs, inducing a triple
cross-talk among populations, maybe creating a specific microenvironment to control
self-renewal and undifferentiated state of melanoma stem cells and thereby fostering
melanoma aggressive potential and treatment failure. Future study combining β-
blockers with common therapies should therefore have important implications for
treatment of malignant melanoma and its total eradication, counteracting at multiple
stages this complex and continuative, catecholamines-mediated interplay between
cancer cells and their microenvironment.
149
EXPERIMENTAL
PART II
150
AIM OF THE STUDY II
In 1926 Otto Warburg demonstrated that cancer cells undergo a glicolytic metabolism
even in presence of oxygen, thereby metabolizing high amounts of glucose into lactate
(Warburg effect), but just recently metabolic reprogramming towards a Warburg
metabolism has been considered as a real hallmark of cancer. Indeed, several studies
have now established that this metabolic shift is crucial for proliferating cells that
require, in addition to ATP, macromolecules in order to sustain cancer progression. In
addition, it has been recently proposed a new metabolic model where cancer cells
induce the Warburg effect in stromal fibroblasts, which undergo a myofibroblastic
differentiation, and secrete lactate and pyruvate in the extracellular space through the
monocarboxylate transporter 4 (MCT-4). Cancer cells then take up these metabolites
through the monocarboxylate transporter-1 (MCT-1) and use them in the mitochondrial
TCA cycle, promoting energy generation via oxidative phosphorylation.
In particular, the aim of this study is to start focusing on the ability of nutrients to
regulate transcription factors involved in cancer cells metabolism. One of these factors
is represented by hypoxia inducible factor-1α (HIF-1α). It is well established that, under
hypoxic condition, HIF-1α activates target genes whose protein products mediate a
switch from oxidative to glycolytic metabolism (ex. GLUT-1, LDH-A), while it is not
well established yet the role of HIF-1α in regulating these complex pathways under
normoxic condition. To this purpose, A375 primary melanoma cell line was cultured in
normoxic condition in presence/absence of nutrients like glucose, pyruvate and lactate
in order to understand how different nutrients can activate key pathways for cancer
progression. Cytofluorimetric analysis demonstrate that A375 show strong dependance
on glucose content of media in order to sustain survival, thereby according to a Warburg
metabolic phenotype, and that nutrients alone are able to induce the expression of HIF-
1α and of its target gene carbonic anidrase-IX (CA-IX), probably thorugh an increased
reactive oxygen species (ROS) production. In order to further understand the role of
nutrients-induced HIF-1α, we therefore performed migration and invasion assays with
Boyden chambers, respectively in absence or presence of Matrigel mimicking the
extracellular matrix. Nutrients alone are also able to elicit a pro-invasive response of
A375 and this effect is reverted after both treatment with Topotecan, a HIF-1α
farmacological inhibitor, and HIF-1α gene silencing. Finally, data obtained so far allow
151
us to hypothesize a novel important role of nutrients-induced HIF-1α normoxic
stabilization in order to sustain cancer progression.
152
RESULTS II
Role of nutrients in regulating melanoma cells proliferation and survival
Today, metabolism reprogram is universally accepted as one of the hallmarks of cancer.
In fact, cancer cells are able to completely modify their metabolic behaviour in order to
sustain cancer progression. Rising evidence have now established that cancer cells
undergo a specific metabolic reprogramming, leading cancer cells to gain several
advantages that are not only related to mere ATP production.
Non-neoplastic cells usually metabolize glucose into pyruvate, which is in turn moved
in the mitochondria, thereby entering the Krebs cycle and undergoing oxidative
phosphorilation. This metabolic pathway produces high ATP levels, quantified in 38
molecules for each glucose molecule, but needs O2 to support mitochondrial respiration.
On the other hand, Otto Warburg demonstrated that cancer cells undergo a glicolytic
metabolism, named “Warburg Effect”, even in presence of oxygen, thereby
metabolizing higher amounts of glucose into lactate, which is then extruded in the
extracellular space through monocarboxylate transporter-1 (MCT-1) (Vander Heiden et
al., 2009). Glucose fermentation does not require oxygen, but it is far less efficient than
the TCA cycle coupled to oxidative phosphorylation in generating ATP. Despite
decreased efficiency in ATP production, fast-growing cells rely primarily on glucose
fermentation during proliferation regardless of oxygen availability. Indeed, several
studies have now established that the Warburg effect, coupled with increased glucose
uptake due to incomplete glucose oxidation, promotes in cancer, high-proliferating cells
the efficient anabolism of macromolecules from glicolytic intermediates, providing the
key carbon precursors needed for the synthesis of nucleic acids, phospholipids, fatty
acids, cholesterol and porphyrins, needed to sustain uncontrolled and continuative
cellular mitosis (Lunt and Vander Heiden, 2011; Pedersen, 2007).
Since the metastatic ability of cancer cells is strongly related to microenvironmental
conditions like nutrients availability, stromal cells and vascularization, their cellular
metabolic fluxes and nutrient demand may show considerable differences. Moreover,
their stage-dependent metastatic ability may further create metabolic alterations
depending on its microenvironment. Although recent studies have aimed to elucidate
cancer cell metabolism in different cancer models, in most of the cases the nutrient
demand and metabolic activity of tumour cells still remains poorly understood due to
their high heterogeneity.
153
For what the melanoma is concerned, the glycolytic phenotype of some melanoma cell
lines has been only recently identified by metabolic profiling using isotopically labeled
nutrients (Scott et al., 2011).
In order to further elucidate the metabolic behavior of melanoma cells, we started
performing crystal violet assay on A375 primary melanoma cell line cultured in
normoxic condition for 24, 48 and 72 hours in total absence (black line) or presence of
nutrients like glucose (red line), pyruvate (green line) and lactate (light blue line). As
culture control condition we cultured melanoma cells in presence of both serum and
glucose (violet line). This assay is useful in order to obtain quantitative information
about the number of cells adhering to multi-well cluster dishes. The dye in this assay,
crystal violet, stains DNA. Upon solubilization, the amount of dye taken up by cultured
cells can be quantitated in a spectrophotometer at 595 nm wavelength. The graph in
Figure 1 shows normalized data as ratio between crystal violet absorbance (nm) and
protein content, thereby allowing us to correlate with the nuclear DNA content and thus
with cell number.
Data obtained demonstrate that nutrients alone are not able to sustain cancer cells
proliferative ability. In fact, A375 cells proliferation rate in presence of glucose,
pyruvate or lactate alone does not show significant modifications if compared with cells
maintained for 24 hours in absence of nutrients while, as expected, the optimal culture
condition represented by both serum and glucose induced a four-fold increase in cells
proliferation rate (Figure 1).
Figure 1. Crystal violet assay on A375 melanoma cells treated for 24, 48 and 72 h with glucose (4,5 g/L),
pyruvate (10 mM) or lactate (10 mM). Culture condition in presence of glucose and serum was
considered as standard control condition. Data are represented as crystal violet Abs (nm)/protein content
ratio and each measurement was made in triplicate for each point of the curve of growth. Experiment was
repeated at least 3 times. p value < 0,005.
0
0,5
1
1,5
2
2,5
3
3,5
4
t0 24h 48h 72h
Cry
stal
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m)/
pro
tein
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nte
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No nutrients
Glucose
Glucose + serum
Pyruvate
Lactate
154
To further elucidate the effect of the different nutrients in cancer cells survival, we
therefore performed an Annexin V-iodidium propide staining assay in order to evaluate
apoptotic cells percentage. Annexin-V is a Ca2+
dependent phospholipid-binding protein
that has high affinity for phosphatidylserine, which itself is translocated from the inner
to the outer leaflet of the plasma membrane during the early phase of the apoptotic
process. Apoptotic cells were identified by flow cytometry using Annexin-V conjugated
to fluorescein isothiocyanate (FITC), in conjunction with propidium iodide (PI) to
distinguish apoptotic cells (Annexin-V-FITC positive, PI negative) from necrotic cells
(Annexin-V-FITC positive, PI positive).
Using this method as a marker of apoptosis, exposure of melanoma cells to 4,5 g/L
glucose, alone or in combination with serum, under serum-free conditions for 24 h led
to a significant decrease (17.4% compared to 6.4%) in the number of cells in early and
late apoptosis as compared to cells cultured in total nutrients absence for the same time.
In contrast, addition of pyruvate or lactate alone induced melanoma cells apoptosis to
levels comparable to that related to nutrients withdrawal, while only combination of
both nutrients with serum was able to significantly reduce apoptotic cells percentage
(Figure 2).
155
Figure 2. Annexin V/Iodidium Propide cytofluorimetric staining on A375 melanoma cells treated for 24h
with glucose (4,5 g/L), pyruvate (10 mM) or lactate (10 mM), alone or in combination with serum. Cells
were then pelletted and labeled with annexin V and iodidium propide. Finally cells were evaluated by
flow citometry. Data are represented as total percentage of cells positive to annexin V staining alone
(early apoptosis) and together with PI (late apoptosis). Experiment was repeated at least 3 times. p value
< 0,005.
To further elucidate the molecular mechanisms underlying nutrients-based cell survival,
we therefore evaluated the possible role of nutrients in directly influencing Akt
activation. Akt is a central player in processes downstream of activated growth factor
receptor signaling like the insulin receptor, EGF-R and HGF-R regulating cells
proliferation and survival. After ligand binding and subsequent receptor activation, Akt
recruitment to the plasma membrane is mediated mainly through phosphatidylinositol 3-
kinase (PI3K), which phosphorylates phosphoinositides to generate
phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Akt amino terminal pleckstrin
homology (PH) domain binds PIP3, thereby promoting the translocation of Akt to the
plasma membrane where it is phosphorylated and activated. The tumor suppressor
PTEN acts as a regulator of Akt activity by dephosphorylating PIP3, although it is
frequently downregulated or lost during tumor progression, contributing to deregulation
of the pathway in cancer cells. After activation, Akt is able to translocate to the nucleus
where it affects both directly or indirectly cellular proliferation and survival. Among
Akt substrates there are transcriptional regulators like CREB, E2F, NF-κB, the FOXO
0
5
10
15
20
25
% A
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PTO
TIC
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LS
156
transcription factors and murine double minute 2 protein (MDM2), master regulator of
p53 activity. In addition, Akt is able to target a number of other molecules to affect the
survival state of the cell including the pro-apoptotic molecule Bcl-2-associated death
promoter (BAD) and caspase 9 (Altomare and Khaled, 2012).
In order to evaluate Akt activation, melanoma A375 cells were serum-deprived
overnight and then cultured for 24 h in presence of glucose (4,5 g/L) or pyruvate (10
mM), alone or in combination with serum. As demonstrated by the western blot
represented in figure 3, melanoma cells cultured for 24h in presence of nutrients alone
do not show activation of the Akt-mediated pathway, while the combination of nutrients
and serum in the culture medium induced a strong increase in the level of
phosphorylated protein (Figure 3).
Figure 3. Activation of AKT. Melanoma A375 cells were serum-deprived overnight and then cultured for
24 h in presence of glucose (4,5 g/L) or pyruvate (10 mM), alone or in combination with serum. An
immunoblot analysis for the detection of the phosphorylation level of p-AKT was shown. Total AKT
immunoblot was used for normalization. The western blot is representative of three independent
experiments.
Taken together, these data demonstrate that nutrients alone do not affect significantly
A375 melanoma cells proliferative ability, as confirmed by lacking of Akt activation in
conditions of serum withdrawal. On the other hand, according to a Warburg-like
metabolic phenotype, Annexin V-IP staining assay showed a high dependence of
p-AKT
AKT
Serum - + - + - + - +
Glucose - - + + - - - -
Pyruvate - - - - + + - -
Lactate - - - - - - + +
Glucose
157
melanoma cells from glucose in order to sustain their survival, likely through an Akt-
independent pathway.
158
Nutrients promote normoxic HIF-1α and CA-IX expression and affect ROS
production
Hypoxia is a common features of solid tumours and complexes HIF-1α, HIF-2α and
HIF-3α are the master regulators of cell response to hypoxia. In particular, HIF-1 is an
heterodimeric complex characterized by an α subunit of 120 kDa O2-dependent (HIF-
1α), and a β subunit of 91-94 kDa constitutively expressed (HIF-1β) also known as aryl
hydrocarbon nuclear translocator (ARNT), as it has been first identified inside the
heterodimeric complex with the aryl hydrocarbon receptor (AHR) (Reyes et al., 1992).
In normoxic conditions HIF-1α shows a very short half-life of about 1/2 ~ 5 minutes
and is rapidly degradated via ubiquitin-proteasome system (Salceda et al., 1997). When
cells are exposed to low O2 tensions, HIF-1α half-life is of about 30 minutes, the protein
is stabilized and traslocates to the nucleus, where it binds to subunit HIF-1β forming the
transcriptionally active HIF complex. The resulting heterodimer binds to HRE sequence
of target genes and binds to transcriptional coactivators, thereby promoting gene
expression (Lando et al., 2002). As already stated, HIF-1α directly regulates several
genes coding for enzymes involved in cells metabolism, in particular glycolytic
metabolism, including glucose transporters and glycolytic enzymes like hexokinase 1
and 2 (HK1 e HK2), PFK1, aldolase A and C, glyceraldheyde-3-phosphate
dehydrogenase, phosphoglycerate kinase 1, enolase 1, pyruvate kinase and lactate
dehydrogenase A (LDHA).
We therefore analyzed if melanoma cells exposure to different nutrients can regulate
transcription factors involved in cancer cells metabolism. We focused on the expression
of HIF-1α as it is well established that, under hypoxic condition, HIF-1α is a master
regulator of anaerobic metabolism, while it is not well established yet the role of HIF-1α
in regulating these complex pathways under normoxic conditions.
To this purpose, A375 primary melanoma cell line was nutrients-starved overnight and
then cultured in normoxic condition for 24h in presence/absence of nutrients like
glucose, pyruvate and lactate. Cells were then lysed and a western blot analysis of HIF-
1α expression was performer in order to understand how different nutrients can activate
key pathways for cancer progression. As a control condition cells were cultured for 24h
also in presence of glucose, pyruvate and lactate in combination with serum. In fact,
HIF-1α expression induced by growth factors stimulation is associated to an increased
production through activation of phosphatydil-inositol-3-kinase (PI3K) and MAPK
pathways (Fukuda et al., 2002; Fukuda et al., 2003; Laughner et al., 2001; Zhong et al.,
159
2000). According to that, it has been demonstrated that MAPK pathway leads to
activation of both p42/p44 MAPK and p38 MAPK, following previous activation of
proteins Ras/Raf-1/MEK-1. It has been demonstrated that HIF-1α is phosphorilated by
p42, p44, p38α and p38γ in vitro (Richard et al 1999; Sodhi et al 2000).
As shown in figure 4, all nutrients alone are able to promote normoxic HIF-1α
stabilization at levels comparable to HIF-1α expression in melanoma cells cultured in
presence of nutrients in combination with serum. In particular, glucose shows higher
effectiveness in inducing HIF-1α expression.
We therefore evaluated the expression of HIF-1α main target gene, carbonic anidrase IX
(CA-IX). Again, western blot analysis shows that nutrients alone, and in particular
glucose, are able to promote also normoxic CA-IX expression (figure 4).
Figure 4. Evaluation of HIF-1α and CA-IX expression. Melanoma A375 cells were serum-deprived
overnight and then cultured for 24 h in presence of glucose (4,5 g/L), pyruvate (10 mM) or lactate (10
mM), alone or in combination with serum. An immunoblot analysis for the detection of HIF-1α and CA-
IX was shown. Actin immunoblot was used for normalization.
In the light of these data, we next analyzed the level of ROS produced by melanoma
cells in presence of different metabolites in order to rely their role to normoxic
nutrients-mediated HIF-1α expression. In fact, it has been already demonstrated that
ROS can act as second messengers in several signaling pathways regulating cancer
progression, like epithelial-mesenchymal transition (EMT), migration/invasion and
angiogenesis. In particular, hypoxia is directly related to higher mitochondrial ROS
HIF-1α
CA-IX
Serum - + - + - + - +
Glucose - - + + - - - -
Pyruvate - - - - + + - -
Lactate - - - - - - + +
Glucose
Actin
160
production, thereby promoting HIF-1α stabilization through inhibition of PHDs activity
(Klimova 2008; Wheng-sheng Wu 2006).
To evaluate the amount of ROS produced, staining with 2',7'-
dichlorodihydrofluorescein diacetate (H2DCFDA) probe was performed. H2DCFDA is a
chloromethyl derivative of H2DCFDA. H2DCFDA passively diffuses into cells, where
its acetate groups are cleaved by intracellular esterases and its thiol-reactive
chloromethyl group reacts with intracellular glutathione and other thiols. The assay was
performed on A375 cells serum-starved overnight and then cultured in normoxic
conditions for additional 24 hours in presence of glucose, pyruvate and lactate, alone or
in combination with serum. Analysis of H2DCFDA absorbance revealed that melanoma
cells show significantly higher production of ROS after culture in presence of
metabolites alone, which is in agreement with the nutrients-induced HIF-1α
stabilization. Again, culture of melanoma cells with glucose alone induced the highest
spike of ROS production, according to its role in promoting highest HIF-1α protein
expression (figure 5).
Figure 5. Intracellular ROS amount evaluation using fluorimetric probe H2DCFDA. A375 melanoma
cells were serum-deprived overnight and then treated for 24 h with glucose (4,5 g/L), pyruvate (10 mM)
or lactate (10 mM), alone or in combination with serum. Then H2DCF-DA (5 g/ml) probe was added
and incubated for 3 minutes at 37°C. After the incubation time, cells were quickly lisated in RIPA lysis
buffer and lysates were then collected, centrifugated, transferred on a 96 wells multiwell and analyzed.
Data are represented as absorbance / protein quantification ratio. Experiment was repeated at least 3
times. p value < 0,005.
0
1
2
3
4
5
6
7
Ab
sorb
ance
/Pro
tein
qu
anti
fica
tio
n
161
Taken together, these data demonstrate that nutrients alone are able to directly regulate
expression of HIF-1α and CA-IX in the human melanoma model. In addition, glucose,
pyruvate and lactate induce high ROS production, thereby allowing us to speculate that
nutrients promote normoxic HIF-1α stabilization in a redox-dependent manner.
162
Nutrients promote CA-IX expression in a HIF-1α-dependent manner
Carbonic anhydrase IX (CA-IX) is a highly active, tumor-associated transmembrane
carbonic anhydrase isoform composed by an extracellular catalytic domain, whose
activity is hypoxia-dependent. In fact, CA-IX expression pattern is strictly related to
HIF-1α-mediated transcriptional activation of CA9 gene, as CA9 promoter region
contains several HRE elements that induce CA-IX expression in response to hypoxia
(Kaluz et al., 2002).
The most important CA-IX role is represented by hydration of pericellular CO2. This
reaction generates an extracellular proton, which contributes to generating an
increasingly acidic extracellular pH, facilitating tumor cell invasiveness, and
bicarbonate ion, which is delivered to cytoplasm through the bicarbonate exchangers
directly interacting with CA-IX in order to maintain an alkyline intracellular pH
favorable for tumor growth (Svastova et al., 2004). In addition to its role in the
regulation of tumoral pH and tumor cell survival, there is evidence that CA-IX
contributes to cell processes such as adhesion and migration, both of which are crucial
for metastatic progression in human cancer (McDonald et al., 2012).
To further confirm the involvement of HIF-1α in the normoxic, nutrients-based
induction of CA-IX expression, we performed specific experiments of inhibition of
HIF-1α expression. We first examined HIF-1α expression levels in melanoma cells
stimulated with glucose, pyruvate and lactate for 24 h, alone or in combination with the
camptothecin analogue Topotecan, a topoisomerase I pharmacological poison which has
been already demonstrated as an HIF-1α protein accumulation blocking agent
(Rapisarda et a., 2004).
Western blot analysis shown in figure 6 clearly demonstrates that melanoma cells
treatment for 24 h with Topotecan impairs normoxic nutrients-induced HIF-1α
expression. Consistent with this finding, Topotecan significantly reduced CA-IX
accumulation as well (figure 6).
163
Figure 6. Evaluation of HIF-1α and CA-IX expression in absence or presence of Topotecan 250 nM.
Melanoma A375 cells were serum-deprived overnight and then cultured in presence of glucose (4,5 g/L),
pyruvate (10 mM) or lactate (10 mM) and stimulated or not with Topotecan for 24 h. An immunoblot
analysis for the detection of HIF-1α and CA-IX was shown. Actin immunoblot was used for
normalization.
To further confirm the results obtained so far we then used a specific siRNA targeting
HIF-1α mRNA for knockdown of gene expression. Again, HIF-1α knockdown cells
resulted in almost total abrogation of HIF-1α and CA-IX protein accumulation,
according to siRNA highest efficiency in downregulating protein expression if
compared to pharmacological treatment (figure 7).
HIF-1α
CA-IX
Actin
+ Topotecan 250 mM
No
nu
trie
nts
Glu
cose
Py
ruv
ate
La
cta
te
Ser
um
No
nu
trie
nts
Glu
cose
Py
ruv
ate
La
cta
te
Ser
um
164
Figure 7. Evaluation of HIF-1α and CA-IX expression after HIF-1α gene silencing. Melanoma A375
cells were silenced for HIF-1α as previously described and then cultured in presence of glucose (4,5 g/L),
pyruvate (10 mM) or lactate (10 mM) for 24 h. An immunoblot analysis for the detection of HIF-1α and
CA-IX was shown. Actin immunoblot was used for normalization. p < 0,05.
HIF-1α
CA-IX
Actin
HIF-1α -
No
nu
trie
nts
Glu
cose
Py
ruv
ate
La
cta
te
Ser
um
No
nu
trie
nts
Glu
cose
Py
ruv
ate
La
cta
te
Ser
um
165
Nutrients-induced HIF-1α expression promotes melanoma cells invasiveness
We therefore evaluated the role of nutrients-induced HIF-1α expression in regulating
cancer cells invasive ability. In fact HIF-1α activation correlates with metastasis in
multiple tumors and can promote metastasis through the regulation of key factors
regulating tumor cell metastatic potential, including E-cadherin, lysyl oxidase (LOX),
CXCR4, and stromal-derived factor 1 (SDF-1) (Rankin and Giaccia, 2008).
To this aim we performed in vitro Boyden assays. The Boyden chamber is composed by
un upper and a lower compartment separated by a microporous membrane with pores of
8 μm of diameter. In order to evaluate cancer cells ability to degrade and invade through
an organic matrix, the Boyden chamber microporous membranes were covered with
Matrigel, a syntethic preparation rich in laminin, collagen IV and entactin and heparan
sulfate proteoglycan (perlecan). This matrix acts as a reconstituted basement membrane
in vitro, occluding the pores of the membrane and blocking non-invasive cells from
migrating through the membrane, while invasive cells can secrete proteases that
enzymatically degrade the Matrigel matrix and enable invasion through the membrane
pores.
For our experiments, A375 were plated in the upper chamber in 200 μl of serum-free
medium in absence or presence of serum, glucose, pyruvate or lactate, while the lower
compartment was filled with 500 culture μl of medium with 10% FBS as a standard
chemotactic agent. Cells were then allowed to migrate through the pores to the other
side of the membrane and, after a 24h incubation time in normoxic conditions, they
were stained with haematoxylin-eosin staining. Finally, the number of cells that had
migrated to the lower side of the membrane was determined by image capture of cells
attached through a microscope and then counting the number of cells invaded to the
lower membrane surface.
As shown in both figures 8 and 9, all nutrients increase the invasive spur of melanoma
cells, with glucose greatly increasing A375 cells invasive ability. In addition, the pro-
invasive effect of all nutrients is strongly sensitive to both Topotecan treatment (figure
8) and HIF-1α gene silencing (figure 9). Again, melanoma cells treatment with
Topotecan resulted in high impairement of A375 motility, while HIF-1α knockdown
cells showed almost total abrogation of invasive ability, in keeping with the results
obtained from western blot analysis (figures 6 and 7).
166
Figure 8. Evaluation of A375 invasive ability through Boyden chamber assay. Melanoma A375 cells
were treated as previously described and then loaded into the upper compartment of the Boyden chamber
in serum-free growth medium, with or without 250 mM Topotecan. After 24 h of incubation at 37°C, the
microporous membrane was fixed in 96% methanol and stained with Diff-Quick solution. The bar graph
shows the number of migrated cells to the lower surface of the polycarbonate filters from three
independent experiments. P value < 0,05.
Untreated
Pyruvate
Lactate
No nutrients
+ Topotecan 250 mM
Glucose
Serum
0
200
400
600
800
1000
1200
Nu
mb
er
of
inva
de
d c
ells
+ Topotecan 250 mM
167
Figure 9. Evaluation of A375 invasive ability through Boyden chamber assay. Melanoma A375 cells
were silenced for HIF-1α as previously described and then loaded into the upper compartment of the
Boyden chamber. After 24 h of incubation at 37°C, the microporous membrane was fixed in 96%
methanol and stained with Diff-Quick solution. The bar graph shows the number of invaded cells to the
lower surface of the polycarbonate filters from three independent experiments. P value < 0,05.
Ctrl
Pyruvate
Lactate
No nutrients
HIF-1α -
Glucose
Serum
0
100
200
300
400
500
600
700
800
900
1000
Nu
mb
er
of
inva
de
d c
ells
HIF-1α -
168
In conclusion, data obtained so far indicate that nutrients are able to directly regulate
HIF-1α and CA-IX expression in normoxic conditions, and it is likely that this
stabilization is redox-dependent. In addition, experiments performed using siRNA
targeting HIF-1α and Topotecan treatment demonstrate that melanoma cells exploit
nutrients by increasing invasiveness through a HIF-1α-dependent mechanism.
169
DISCUSSION II
The urgent need of always new antineoplastic drugs is at present a real challenge for
molecular oncologists. In particular, the recently renewed interest for the metabolic
deregulation of tumors has led to new pharmacological strategies targeting metabolic
pathways to impair cancer cells survival. In fact, approximately 60% to 90% of cancers
display a Warburg-like metabolic profile that is, the capacity of non-hypoxic tumor cells
to entirely rely on glycolysis without exploiting oxidative phosphorylation in order to
support the high biosynthetic and energy demands of actively proliferating cells.
Therefore, tumours characterized by aerobic glycolysis and/or high glucose dependence
could be more sensitive than other tumours to agents targeting metabolic-related key
cancer pathways.
Metastatic melanoma represents a very aggressive tumour, which relays to more than
the 80% of total deaths caused by skin cancers, thanks to its intrinsic resistance to
common pharmacological therapies (Helmback et al., 2001). Just recently a report by
Scott and collegues characterized the metabolic profile of several melanoma cell lines,
showing that cancer cells all exhibited a Warburg metabolism with higher glucose
consumption rate and lactate production compared to healthy melanocytes (Scott et al.,
2011).
According to these observations, our data indicate that metastatic melanoma cells A375
display a high dependence from glucose, as the Annexin V/IP staining assay
demonstrated that only glucose administration was able to significantly reduce the
percentage of apoptotic cells. As a further confirmation, pyruvate alone was not able to
sustain melanoma cells survival, thereby allowing us to hypothesize reduced TCA cycle
and oxidative phosphorylation activity in favor of a Warburg metabolism. On the other
hand, we observed that glucose, pyruvate and lactate are not able to elicit melanoma
cells proliferation, as confirmed also by the western blot analysis on Akt
phosphorylation levels.
Additionally, we report that increased aggressiveness of human metastatic melanoma
A375 cells is tightly correlated to a normoxic, nutrients-induced HIF-1α stabilization.
Our finding of HIF-1α acting as a nutrients-responsive transcription factor unravels a
pathway independent from the O2 extracellular concentration which may connect tumor
cell metabolism and cancer cells motility. These data are proposed to further support the
importance of Warburg metabolism for aggressive tumour cells. According to that, as
170
many genes coding for glycolytic enzymes, glucose transporters, and glucose regulatory
hormones are induced by hypoxia (Dang and Semenza, 1999; Feldser et al., 1999), the
Warburg effect may represent a feed-forward mechanism to maintain a high
intracellular concentration of metabolites and intermediates in order to foster the
expression of HIF-1α-related genes even in normoxic condition. In addition, in our
experimental model all nutrients promoted high normoxic ROS accumulation. Previous
studies by Lu and colleagues reported that several TCA cycle intermediates can stabilize
HIF-1α through inhibition of PHD enzymes (Lu et al., 2002; Lu et al., 2005). Therefore,
it is highly conceivable that in our experimental model nutrients-based ROS delivery
during normoxia could play an important role in promoting HIF-1α expression through
inactivation of PHDs activity.
In addition, our data also indicate HIF-1α as the driver of a nutrient-dependent pathway
mediating melanoma motility. This conclusion was supported by experiments
performed using both Topotecan, a known HIF-1α pharmacological inhibitor, and a
specific siRNA targeting HIF-1α, which almost completely blocked nutrients-induced
cancer cells invasiveness. Our data are in agreement with a recent report by Goetze and
collegues who observed that both pyruvate and lactate have been shown to increase the
migration of head and neck cancer cells (Goetze et al., 2011). However, to our
knowledge, our study is the first to show that nutrients have the potential to increase the
invasive ability and metastatic potential of aggressive melanoma cells through an HIF-
1α-dependent pathway also in normoxic condition. HIF-1α has already been proposed
as a key player of cancer cells invasiveness, and mechanisms proposed are mainly
correlated to its ability to regulate key factors governing tumor cell metastatic potential,
including E-cadherin, lysyl oxidase (LOX), CXCR4, and stromal-derived factor 1
(SDF-1) (Rankin and Giaccia, 2008). We also recently reported that aggressive
melanoma cells respond to hypoxia upregulating their motile/invasive abilities, based on
redox stabilization of HIF-1α and activation of the Met protoncogene, allowing a
proteolytic motility enhancing metastatic dissemination to lungs (Comito et al., 2011). It
is then conceivable that lack of proliferation in presence of nutrients alone could be seen
as a strategy leading to protection against a condition of metabolic stress, thereby
favouring cancer cells motogen escaping strategy. Furthermore, also HIF-1α has been
recently described as a negative regulator of cancer cells proliferation through activation
of different mechanisms. For example, it has been reported that HIF-1α promotes cell
cycle arrest through induction of p27 overexpression regardless of hypoxic conditions
171
(Hackenbeck et al., 2009), and Kaidi and collegues demonstrated that HIF-1α induces
overexpression of the main cyclin-dependent kinase (CDK) inhibitors p21 and
p27 by antagonizing c-Myc transcriptional activity, thereby leading to cell cycle arrest
into G1 phase (Kaidi et al., 2007).
In the light of these observations, we can speculate that also nutrients-dependent
carbonic anidrase IX (CA-IX) expression could contribute to enhanced melanoma
aggressiveness. CA-IX overexpression has been demonstrated in several cancer models
as a master regulator of pH homeostasis, which is crucial for cancer cells in order to
avoid potentially harmful effects of an highly glycolytic and therefore pro-acidic
metabolism (Robertson et al., 2004; Neri and Supuran 2011). For this reason, several
studies have started clarifying the role of CA-IX in eliciting cells motility. Initially
Parkila and collegues demonstrated that acetazolamide, a potent specific CA inhibitor,
reduced renal cancer cells invasive ability in vitro (Parkkila et al., 2000). More recently,
in colorectal cancer cells it has been proved that COX-2-dependent expression of CA-
IX correlates with tumour stage and increases cancer cells invasiveness (Sansone et al.,
2008). Additionally, Svastova et collegues pointed out the ability of CA-IX to modulate
E-cadherin-dependent cell adhesion through direct colocalization with β-catenin and to
promote loss of cell-cell contact, the key initial step of cancer invasion (Svastova et al.,
2003). Therefore, gene silencing experiments will be crucial to completely elucidate the
ability of nutrients to possibly regulate melanoma cells invasiveness through a CA-IX-
dependent mechanism.
In conclusion, our data propose the normoxic, nutrients-and redox-induced HIF-1α as a
mandatory player in melanoma aggressiveness, acting as a promoter of melanoma cells
invasiveness. In this light, future studies aimed on targeting specific metabolic pathways
could therefore have important implications for treatment of malignant melanoma.
172
CONCLUDING REMARKS II
On the basis of the results obtained we can speculate that also protein kinase M2
(PKM2) expression could be directly affected by nutrients, thereby suggesting a
positive feedback mechanism of HIF-1α activation in order to further promote a
Warburg-like metabolic behavior in aggressive melanoma cells. In particular, Pro403
and Pro408 hydroxylation on PKM2 by prolyl hydroxylase-3 (PHD-3) promotes PKM2
transition from tetrameric to dimeric form, which migrates in the nucleus and acts as
HIF-1α direct coactivator. In turn, there is an increased transcription of HIF target genes
like PKM2 and EGLN3, encoding for PKM2 and PHD3, respectively, thereby engaging
a feed-forward loop leading to amplified HIF-1α activity (Semenza et al., 2011).
Another fascinating hypothesis is that metabolites in the diffusion-limited tumor
microenvironment could be acting as paracrine signaling molecules. For example, it has
been recently demonstrated that tumour-secreted lactate can act as a paracrine molecule
able to favour motility in vitro of human mesenchymal stem cells (hMSC) (Rattigan et
al., 2011). We can therefore speculate that nutrients could have a dramatic effect on
tumour progression acting both on cancer cells, promoting HIF-1α-dependent
invasiveness, and on the surrounding stroma, maybe favouring recruitment of
neighbouring stromal cells like cancer-associated fibroblasts (CAFs), thus activating
further pro-metastatic cross-talks. In particular, as we report that glucose, pyruvate and
lactate induce normoxic HIF-1α stabilization, likely through a redox-dependent
mechanism, it is conceivable that nutrients delivered to microenvironment by growing
tumour vasculature could directly modulate stromal cells metabolism, promoting
Warburg metabolism through normoxic stabilization of HIF-1α and favouring metabolic
reprogramming of melanoma cells towards a reverse Warburg phenotype in conditions
of low glucose concentration. At this purpose, we recently demonstrated a metabolic
cross-talk in which PCa cells elicit an HIF-1α and redox-dependent Warburg
metabolism in CAFs, which produce and extrude lactate in turn uploaded by PCa cells
in order to foster their own anabolic pathways and ensure cancer cell proliferation
(Fiaschi et al., 2012). This adaptative mechanism engaged by cancer cells demonstrates
their high metabolic plasticity, as these cells can either use Warburg metabolism in high
glucose environment, but are able to quickly change their metabolic behaviour to a
reverse Warburg programme after CAFs contact during glucose starvation. Finally,
according to this observation, we can speculate that this hypothetical nutrients-driven
173
metabolic liaison between cancer cells and CAFs could involve recruitment and
activation of other key components of tumour reactive stroma, like macrophages and
endothelial cells. Accordingly, lactate has been described to directly modulate cell
signaling networks in endothelial cells upon uptake through the monocarboxylate
transporter-1 (MCT-1), stimulating an autocrine NF-kB/IL-8 pathway driving cell
migration and new vessels formation (Vegran et al., 2011). Moreover, lactate has also a
role in directly promoting macrophage activation and production of proinflammatory
molecules such as IL-6 (Samuvel et al., 2009). It is therefore conceivable that nutrients
can directly modulate metabolic-related intracellular signaling pathways of different
stromal cells in order to foster this complex metabolites-mediated interplay, offering a
both structural and metabolic support essential for tumour survival/growth and
resistance to common therapeutic agents.
174
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APPENDIX:
PUBLICATIONS
b-adrenoceptors are upregulated in human melanomaand their activation releases pro-tumorigenic cytokinesand metalloproteases in melanoma cell linesSilvia Moretti1, Daniela Massi2, Valentina Farini3, Gianna Baroni2, Matteo Parri3, Stefania Innocenti4,Roberto Cecchi5 and Paola Chiarugi3
Recent studies sight b-adrenergic receptor (AR) antagonists as novel therapeutic agents for melanoma, as they mayreduce disease progression. Here within, we evaluated the expression of b-ARs in a series of human cutaneous mela-nocytic lesions, and studied the effect of their endogenous agonists, norepinephrine (NE) and epinephrine (E), on primaryand metastatic human melanoma cell lines. Using immunohistochemistry, we found that both b1- and b2-ARs areexpressed in tissues from benign melanocytic naevi, atypical naevi and malignant melanomas and that expression wassignificantly higher in malignant tumours. Melanoma cell lines (human A375 primary melanoma cell line and humanHs29-4T metastatic melanoma cell lines) also expressed b1- and b2-ARs by measuring transcripts and proteins. NE or Eincreased metalloprotease-dependent motility, released interleukin-6 and 8 (IL-6, IL-8) and vascular endothelial growthfactor (VEGF). These effects of catecholamines were inhibited by the unselective b-AR antagonist propranolol. The role ofsoluble factors elicited by catecholamines seemed pleiotropic as VEGF synergized with NE increased melanoma inva-siveness through 3D barriers, while IL-6 participated in stromal fibroblast activation towards a myofibroblastic phenotype.Our results indicate that NE and E produce in vitro via b-ARs activation a number of biological responses that may exert apro-tumorigenic effect in melanoma cell lines. The observation that b-ARs are upregulated in malignant melanomatissues support the hypothesis that circulating catecholamines NE and E, by activating their receptors, favour melanomaprogression in vivo.Laboratory Investigation (2013) 0, 000–000. doi:10.1038/labinvest.2012.175
KEYWORDS: b-adrenoceptors; immunohistochemistry; melanoma; melanoma progression; norepinephrine/epinephrine
Melanoma represents the most aggressive type of skin cancer,with an increasing incidence found especially in youngadults. A significant reduction in mortality has been notobserved, despite a noteworthy improvement in early diag-nosis achieved in recent years.1 At present, no medical optioncan cure metastatic melanoma (MM) and the only effectivetreatment for the eradication of the disease is early-phasesurgery.2 Hence, increased knowledge of the biologicalpathways underlying the process of melanomadissemination and metastasis is crucial in order to identifynew therapeutic targets.
Previous studies have shown that various human solid tu-mours, such as breast, colon, prostatic, ovary, nasopharyngeal
and oral cancer, express b2-adrenoceptor (b2-AR), raising thepossibility that such receptors may affect invasion and dis-semination processes.3–8 Moreover, some stressneurotransmitters, such as norepinephrine (NE) andepinephrine (E), have been demonstrated to contribute tothe regulation of tumour cell invasion, at least in part throughb-AR activation.6,7,9 Interactions between tumour cells andsoluble factors originated from the nervous system hasrecently been proposed to favour metastasis formation.10
Improved survival rates have been demonstrated in micewith metastatic tumour by combined administration of b-ARantagonists.11 In addition, recent evidence suggests a dramaticrole of b-AR blockers in reducing metastases, tumour
Q1
1Section of Clinical, Preventive and Oncologic Dermatology, Department of Critical Care Medicine and Surgery, University of Florence, Florence, Italy; 2Department ofCritical Care Medicine and Surgery, Division of Pathological Anatomy, University of Florence, Florence, Italy; 3Department of Biochemical Sciences, University ofFlorence, Florence, Italy; 4Division of Pathology, Pistoia Hospital, Pistoia, Italy and 5Dermatology Unit, Pistoia Hospital, Pistoia, ItalyCorrespondence: Professor S Moretti, MD, Section of Clinical, Preventive and Oncological Dermatology, Department of Critical Care Medicine and Surgery, University ofFlorence, Villa S. Chiara, Piazza Indipendenza 11, Florence 50129, Italy.E-mail: [email protected]
Received 11 May 2012; revised 1 December 2012; accepted 2 December 2012
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& 2013 USCAP, Inc All rights reserved 0023-6837/13 $32.00
recurrence and specific mortality in breast cancer patients.12
More recently, the use of b-blockers for concomitant diseasewas associated with a reduced risk of progression of thickmelanoma13 and with an increased survival time of melanomapatients,14 suggesting that the interaction of catecholamineswith b-ARs could be a useful target in this disease.
Expression of b-ARs has been found in melanoma cell linesand in human melanoma biopsies and NE was demonstratedto enhance cytokine production from melanoma cells.15
However, no detailed information regarding b-ARsexpression in human cutaneous benign and malignantmelanocytic lesions or catecholamine influence onmelanoma cell migration has been provided so far. Ouraim was to evaluate the expression of b-ARs on a series ofhuman cutaneous melanocytic naevi and malignantmelanoma, and assess the potential influence of NE and Eon the malignant behaviour of human melanoma cell lines.We could demonstrate a significant upregulation of b-ARsexpression in melanoma in vivo and the activation of pro-tumorigenic biological responses induced by NE and Ein vitro.
MATERIALS AND METHODSHistologic SamplesForty human cutaneous melanocytic lesions from 40 differ-ent patients were evaluated. Tissue samples were retrievedfrom the archives of the Division of Pathological Anatomy,Department of Critical Care Medicine and Surgery, Uni-versity of Florence, Florence, and from the Division of Pa-thology, Pistoia Hospital, Pistoia, Italy.
The study series included five common melanocytic naevi(CN) (two females, three males, age 28–54 years, mean 35.8years); five atypical (so-called dysplastic) melanocytic naevi(AN) (two females, three males, age 30–47 years, mean 40.4years); five in situ primary melanoma (PM) (two females,three males, age 37–55 years, mean 44.2 years; site: threetrunk, one lower extremity); nine superficial spreading (SS)PM (seven females, two males, age 41–82 years, mean 58.4years; site: four trunk, three leg, two arm; thickness 0.30–1.90 mm, mean 0.82 mm; five level II, three level III); sixnodular (N) PM (three females, three males, age 53–76 years,mean 61.5 years; site: three trunk, one leg, two arm; thickness1.40–17 mm, mean 5.2 mm; two level III, three level IV, onelevel V), ten MM, five cutaneous and five lymph-nodal (onefemale, nine males, age 59–87 years, mean 77.1 years).
MaterialsRabbit polyclonal anti-b1- or anti-b2-AR were obtained fromSanta Cruz Biotechnology (Santa Cruz, CA, USA) or Che-micon (Temecula, CA, USA), strepatdvidin-biotin peroxidasecomplex from Ultravision (LabVision, Fremont, CA, USA),DAKO EnVision System HRP from Dako (Carpenteria, CA,USA). For in vitro experiments, unless specified allreagents were obtained from Sigma (St Louis, MO) exceptPVDF membrane (Millipore, Bedford, MA); Matrigel
(BD Biosciences, Bedford, MA); Diff-Quik staining kit(Medion Diagnostics, Miami, FL); Transwell (Corning In-corporated, Corning, NY); ilomastat (Chemicon Interna-tional, Bedford, MA); p38 MAPK (mitogen-activated proteinkinase), phospho-p38 MAPK (Thr180/Tyr182), p44/p42MAPK and phospho-p44/p42 (T202/Y204) monoclonal an-tibodies (Cell Signalling, Danvers, MA). The Amplite TMUniversal Fluorimetric matrix metalloproteinase (MMP)Activity Assay Kit-Red Fluorescence was supplied by AATBioquest, Sunnyvale, CA.
ImmunohistochemistryThe specimens were obtained by surgical resection in all casesand fixed in 10% formalin before being processed in paraffin.Haematoxylin-eosin stained sections from each histologicalspecimen were reviewed to confirm the histological diag-nosis. The protocol was approved by the Institutional ReviewBoard for use of human tissues.
For immunohistochemical analyses, a representative sec-tion of 3 mm for each lesion was selected. All sections weredeparaffined in Bio-Clear (Bio-Optica, Mi, Italy) and hy-drated with grade ethanol concentrations until distilled wa-ter. Antigen retrieval was performed by calibrated water bathcapable of maintaining the Epitope Retrieval Solution EDTA(pH 9.0) at 97 1C for 15 min. The sections were then allowedto cool down to room temperature (RT) for 20 min. To blockendogenous peroxidase activity, slides were treated with 3.0%hydrogen peroxidase in distilled water for 10 min and sub-sequently washed two or three times with PBS. Then poly-clonal antibodies anti-b1- or anti-b2-AR were incubated for1 h at RT at 1:100 dilution or at 1:30 dilution, respectively.Immunohistochemical analysis was performed using thestreptavidin-biotin peroxidase complex for b2-AR, or DAKOEnVision System HRP for b1-AR. Finally, aminoethilcarba-zole (LabVision) was applied for 5 min as chromogen.
Normal eccrine sweat glands intensely express b2-AR, andthis parameter was used as a positive internal control.16
Negative control was performed by substituting the primaryantibody with a non-immune serum at the sameconcentration. The control sections were treated in parallelwith the samples. The sections were lightly counterstainedwith Mayer’s haematoxylin.
Immunostaining was independently assessed by two ob-servers (DM, SM). Discrepancies in the reading were resolvedby a second parallel reading of the slides. The percentageof positive cells per lesion was scored according to semi-quantitative criteria. As the percentage of positive naevusmelanocytes/melanoma cells was always higher than 50%,semi-quantitative results were expressed as score 1 (50–80%positive naevus melanocytes/melanoma cells), score 2(81–90% naevus melanocytes/melanoma cell staining) andscore 3 (91–100% melanoma cell staining). The cell stainingintensity was scored on a scale as weak, moderate, strong,very strong.
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For statistical analysis, non-parametric tests were used todetermine significant differences between groups. The dis-tribution of the scored values of positive lesions after im-munological staining in each group was the unit of analysis.Groups were: naevi, PM, MM. Statistical evaluation wasperformed also comparing CN vs AN, naevi vs malignantlesions, and in situ PM plus SSPM vs NPM and MM. Dif-ferences were assessed using the non-parametric Mann–Whitney U-test for independent samples and were consideredsignificant at Pr0.05.
Cell Lines and Culture ConditionsHuman dermal fibroblasts (HDFs) isolated from a surgicalexplantation taken from healthy patients, human A375 PMcell line and human Hs29-4T MM cell line were cultivated inDMEM supplemented with 10% FCS at 37 1C in a 5% CO2
humidified atmosphere. All experiments were performedwith 70–80% confluent cultures, following 18-h incubationin serum-free culture medium. Cells were then stimulatedwith NE or E, at 1 mM concentration. Where needed, cellswere pre-treated with unselective b-AR antagonist propra-nolol (Sigma-Aldrich) (1 mM). After 1 h, medium was re-moved and cells were stimulated with NE 1 mM with orwithout propranolol 1 mM. For fibroblast activation, cellswere grown to sub-confluence and treated for 24 h with theindicated cytokines. Fresh serum-free medium was added foran additional 24 h before collection of conditioned medium(CM), in order to obtain CM free from cytokines (butconditioned by their earlier administration). HDFs cells werethen incubated with the obtained CM for 24 h and then usedfor western blot analyses.
Western Blot AnalysisCells were lysed for 20 min on ice in 500 ml of complete RIPAlysis buffer (50 mmol/l Tris-HCl (pH 7.5), 150 mmol/l NaCl,1% NP40, 2 mmol/l EGTA, 1 mmol/l sodium orthovanadate,1 mmol/l phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin,10 mg/ml leupeptin). Lysates were clarified by centrifuging,separated by SDS-PAGE and transferred onto nitrocellulose.The immunoblots were incubated in 3% bovine serum al-bumin, 10 mmol/l Tris-HCl (pH 7.5), 1 mmol/l EDTA and0.1% Tween 20 for 1 h at RT and were probed first withspecific antibodies and then with secondary antibodies. Forchemiluminescence detection, we used Gel Logic 2002 KodakImaging System, equipped with a charge-coupled devicecamera, which guarantees high linearity. Quantity One soft-ware (Bio-Rad) was used to obtain quantitative analyses.
Invasion AssayTranswell system, equipped with 8-mm pore poly-vinylpirrolidone-free polycarbonate filters, was used. Cells(5� 104 in 300 ml) were loaded into the upper compartmentin serum-free growth medium with or without 50 mmol/lilomastat. The upper sides of the porous polycarbonate filterswere coated with 50 mg/cm2 of reconstituted Matrigel
basement membrane and placed into six-well culture dishescontaining 1 ml of complete growth medium. After 24 h ofincubation at 37 1C, noninvading cells and the Matrigel layerwere mechanically removed using cotton swabs, and themicroporous membrane was fixed in 96% methanol andstained with Diff-Quick solution. Chemotaxis was evaluatedby counting the cells that migrated to the lower surface of thepolycarbonate filters (six randomly chosen fields,mean±s.d.).
Real-Time PCRTotal RNA was extracted from Hs29-4T and A375 derivedfrom our experimental conditions using the RNeasy Minikitkit. Total RNA (1 mg) was reverse-transcribed using theQuantitect Reverse Transcription Kit. Reverse transcriptionwas performed in a final volume of 20 ml containing reversetranscriptase, real-time buffer 1� and real-time primer mix.The amplification was carried out at 42 1C for 2 min, then42 1C for 15 min and 95 1C for 3 min. Measurement of geneexpression was performed by quantitative real-time PCR(7500 Fast Real-Time PCR System, Applied Biosystems),using the Qiagen Quantifast SYBR Green PCR kit. For eachsample, 1mg of cDNA was added to 25 ml of PCR mix. Thesamples were then subjected to 40 cycles of amplification at95 1C for 10 s and 60 1C for 30 s. RNeasy Minikit, QuantitectReverse Transcription Kit, all primer/probe mixes and QiagenQuantifast SYBR Green PCR Kit were from Qiagen, exceptthe following primers:
ADRB1 FW: 50-CAGGTGAACTCGAAGCCCAC-30
ADRB1 REV: 50-CTCCCATCCCTTCCCAAACT-30
ADRB2 FW: 50-ACGCAGTGCGCTCACCTGCCAGACT-30
ADRB2 REV: 50-GCTCGAACTTGGCAATGGCTGTGA-30
VEGF FW: 50-TACCTCCACCATGCCAAGTG-30
VEGF REV: 50-ATGATTCTCCCTCCTCCTTC-30
IL-8 FW: 50-CTGGCCGTGGCTCTCTTG-30
IL-8 REV: 50-TTAGCACTCCTTGGCAAAACTG-30
MMP-2 FW: 50-ACGACCGCGACAAGAAGTTAT-30
MMP-2 REV: 50-ATTTGTTGCCCAGGAAAGTG-50
(NM_00453; Digestive and liver disease 37 (2005) 584–592)MMP-9 FW: 50-GACAAGCTCTTCGGCTTCTG-30
MMP-9 REV: 50-TCGCTGGTACAGGTCGAGTA-50
IL-6 FW: 50-AGTTCCTGCAGTCCAGCC-30
IL-6 REV: 50-TCAAACTGCATAGCCACTTTC-30
Quantitative MMP Activity AssayMMPs activity was measured with Amplite TM UniversalFluorimetric MMP Activity Assay Kit according to themanufacturer’s instructions. Briefly, serum-free mediumfrom confluent monolayer of cells was collected and 5 ml wereadded to 4-aminophenylmercuric acetate (AMPA; 1 nmol/l)at 37 1C for 1 h to detect MMP-2 activity and at 37 1C for 2 hto detect MMP-9 activity. A 50 ml portion of the mixture wasthen added to 50 ml of MMP Red substrate solution. After60 min of incubation the signal was read by fluorescence
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microplate reader with excitation (Ex)/emission (Em)¼ 540nm/590 nm.
Statistical AnalysisIn vitro data are presented as means±s.d. from at least threeexperiments. Results were normalized vs control expressionlevels. Statistical analysis of the data were performed byStudent’s t-test. Pr0.05 was considered statistically sig-nificant.
RESULTSExpression of b-ARs in Tissue SamplesThe presence of b-ARs was demonstrated in all melanocyticlesions examined. The staining for b1-AR was confined to thecell cytoplasm in naevus melanocytes and melanoma cells;the reaction for b2-AR was also confined to the cell cyto-plasm in all cases, with an additional peripheral membranepattern in some AN melanocytes and malignant cells. The cellstaining intensity was always weak with regard to the reactionfor b1-AR, whereas the immunostaining for b2-AR appearedto be weak in CN, moderate (except one very-strong reac-tion), in AN; moderate or strong in in situ PM; from weak tomoderate or strong in SSPM, and from strong to very strongin NPM and MM, with no difference between cutaneous andnodal metastasis.
The immunostaining of each lesion taking into accountboth reaction intensity and percentage of positive cells isshown in Figure 1. In regards to b1-AR expression(Figure 1a), score 1 was evaluated in both CN and AN, score2 was found in a minority (3 in situ and 2 SS) of PM, whereas
in the other PM and MM score 3 was detected. b1-ARexpression was significantly higher in malignant than in be-nign lesions (Pr0.0001) and in PM or MM than in naevi(Pr0.0001 and Pr0.0001). No difference was observed be-tween CN and AN, or between in situ/SSPM compared withNPM/MM.
With regards to b2-AR expression (Figure 1b), score 1 wasobserved in CN, score 2 in AN and score 3 was detected inall PM and MM but one (SSPM), which exhibited score 2.b2-AR reactivity was significantly higher in malignant lesionsthan in naevi (Pr0.0001), and in PM or MM, respectively,than in naevi (Pr0.0001 and Pr0.0001). AN exhibited asignificantly higher reactivity compared with CN (Pr0.003),and no difference was observed between in situ/SSPM andNPM/MM.
In addition, no significant difference was detected betweenPM and MM for both receptors.
Examples of reactions of melanocytic lesions for b1- andb2-AR are shown in Figure 2. Epidermal keratinocytes werelightly coloured for b2-AR, as previously described.17
Endothelial and stromal cells exhibited heavy reactivity forb2-AR in malignant lesions, and to some extent, in AN.
Taken together, our data showed that b1- and b2-ARs werevariably expressed in human melanocytic lesions with a sig-nificant upregulation in PM and MM, and, at least for b2-AR,a significant upregulation was also observed in AN vs CN.
Effects of Catecholamines on Cancer Cell MotilityIn order to confirm the correlation between sensitivity tocatecholamines and progression towards a malignant phe-
Figure 1 Immunohistochemical expression of b1-AR (a) and b2-AR (b) in cutaneous human melanocytic lesions: percentage of positivity and staining
intensity in each lesion. Each circle represents the percentage of stained cells for one lesion. A quarter-black circle indicates positive weak staining; a
half-black circle indicates positive moderate staining; a three-quarter-black circle indicates positive strong staining; a solid-black circle indicates very-
strong staining.
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notype of melanoma cells, we treated with NE or E twohuman melanoma cell lines, namely Hs29-4T cells, selectedfrom a metastatic lesion, and A375 cells, derived from PM.We observed that both cell lines express low and comparablelevels of b1-AR, as shown in Figure 3, while they both expresshigher amounts of b2-AR (Figures 4a and b), with the pri-mary A375 melanoma cell line exhibiting a significantlyhigher expression of b2-AR compared with the metastatic
Hs29-4T cell line. Both cell lines are able to respond to ca-techolamine stimulation with protein kinase A (PKA)phosphorylation, a known trait of b-AR stimulation (Figures4c and d). In addition, we evaluated the effects of both ca-techolamines on the MAPK pathways, as few studies untilnow have investigated the effects of b-adrenergic signallingon these molecular pathways in melanoma models. As shownin Figure 5, both NE and E are able to induce activation,
Figure 2 Expression of b1-AR and b2-AR in human cutaneous melanocytic lesions. b1-AR immunostaining in CN (a), AN (b), in situ PM (c), SSPM (d),
NPM (e), nodal MM (f): all lesions show a low reaction intensity, confined to the cell cytoplasm. b2-AR immunoreactivity in CN (g), AN (h), in situ PM (i),
SSPM (j), NPM (k), nodal MM (l): except the weak staining intensity observed in CN, a moderate to strong or very-strong reactivity intensity is detected
in all the other lesions. While CN cells show only cytoplasmic staining for b2-AR, AN and melanoma cells show cytoplasmic and membranous positivity;
stromal cells show some reactivity in malignant lesions and AN (Scale bar, 50 mm).
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although with different kinetics, of p42-p44 and p38 MAPKin both cell lines (Figures 5a and b).
We then analysed the 3D motility of these cells upon ca-techolamine stimulation. The invasion assay, carried out byBoyden chambers covered with Matrigel to mimic a 3Dbarrier, revealed that both NE and E are able to elicit invasivebehaviour in both metastatic or PM cells. Both NE and Eappear to be more effective in PM cells with respect to MMcells. In addition, E is the most efficient catecholamine ineliciting invasiveness of the metastatic Hs29-4T cell line(Figures 6a and b). In both cells lines the increase in 3Dinvasiveness is sensible to treatment with ilomastat, a broadrange inhibitor of MMPs (Figures 6a and b). The pro-invasive effect of both NE and E is strongly sensitive topropranolol, thereby confirming the involvement of b sub-types of AR. The last finding suggests the involvement of aproteolytic degradation of the Matrigel barrier during inva-sion. We, therefore, analysed by Real-Time PCR the expres-sion of MMP-2 and MMP-9, the main proteolytc enzymesexpressed by Hs29-4T and A375 cell lines, during stimulationwith catecholamines. Figures 6c and d reveal that, while NEand E do not influence MMP-9 production, the expression ofMMP-2 is increased by NE in A375 PM cells and by E inmetastatic Hs29-4T. More importantly, both NE and E areable to maintain a high activation state of secreted MMP-2and MMP-9 following catecholamines stimulation for 24 hon both cell lines (Figures 6e and f).
Effects of Catecholamines on Cytokine Production andTumour MicroenvironmentCancer cells secrete many cytokines, chemokines and growthfactors that can affect their own aggressiveness, in terms of
proliferation, invasion or survival, as well the reactivity of thesurrounding stroma. To address the role of catecholaminestimulation in these features we first analysed the expressionof a panel of cytokines/growth factors by real-time PCR. Wefound that catecholamine stimulation leads to an increase inthe expression of VEGF, IL-6 and IL-8 (Figures 7a–c). In-terestingly the two catecholamines used show differentialeffects for VEGF, IL-6 and IL-8 in Hs29-4T and A375 cells. InA375 PM cells both NE or E are able to elicit expression ofVEGF, IL-6 and IL-8. Conversely, the Hs29-4T metastatic cellline senses NE to increase expression of IL-6 and IL-8, and Eto express VEGF.
The role of VEGF, IL-6 and IL-8 in tumour progressionvaries from increase in invasiveness/scattering and growth ofangiogenic sprouting for VEGF and IL-8, to activation ofstromal and/or inflammatory cells for IL-6.18–20 We observedthat VEGF stimulation increases the invasive spur induced byNE in A375 PM cells, while IL-6 does not have a role(Figure 8a). On the other side, we analysed the ability of IL-6,in association with NE, to activate dermal fibroblasts. Weobserved that IL-6 is able to activate dermal fibroblasts, asdemonstrated by their ability to express a-smooth muscleactin (a-SMA), an acknowledged marker of myofibro-blasts.21,22 In addition, in dermal fibroblasts, the CM of NE-treated A375 cells elicits an activation state very similar withrespect to treatment with IL-6 alone. Conversely, VEGFtreatment is almost ineffective in eliciting a reactivity offibroblasts (Figure 8b).
Taken together, these data suggest that in vitro the treat-ment of human melanoma cells with catecholamines dra-matically affects their aggressiveness, inducing expression ofMMP-2, VEGF, IL-6 and IL-8. These factors orchestrate afeed-forward loop leading to increase of proteolytic inva-siveness of tumour cells, as well as activating surroundingfibroblasts.
DiscussionThe present study shows that the immunohistochemical ex-pression of b-1 and b-2 ARs is significantly upregulated inmelanoma tissues. Interestingly, all tested melanocytic lesionsexhibited some immunoreactivity, suggesting that both be-nign and malignant lesions can theoretically be affected bycatecholamines in vivo. However, since b-ARs staining sig-nificantly increases in PM and MM compared with mela-nocytic naevi, it appears that malignant lesions can be moredeeply influenced by catecholamines than benign counter-parts. The staining intensity for b2-AR progressively in-creased from CN, towards AN, to PM and MM, whereas thereaction intensity for b1-AR was weaker, in all groups oflesions. Such a difference can rely on a different reactivity ofthe used antibodies, but it is likely that b1-AR is expressed onnaevus melanocytes and melanoma cells of sections at alower level than b2-AR. This hypothesis was supported byPCR and western blot analysis of primary and MM cell lines,
Figure 3 Expression of b1-AR in melanoma cell lines. (a) Analysis of b1-AR
expression by immunoblot in primary (A375) and metastatic (Hs29-4T)
melanoma cell lines. (b) Amount of ADRB1 mRNA by real-time PCR. The
amount of target, normalized to the endogenous reference (18S RNA), was
given by the 2�DDct calculation and was reported as 2�DDct. Both
immunoblots and real-time PCR are the mean of three independent assays.
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both of which exhibited a lower expression of b1-AR vsb2-AR.
In melanoma tissue sections a strong reactivity for b2-ARwas also observed in most endothelial and stromal cells, in-cluding macrophages, suggesting the possible influence ofcatecholamines on cells of the tumour microenvironmentand the chance of further potential biologic loops capable ofaffecting metastatic behaviour of neoplastic cells.
We also demonstrated that A375 primary and Hs29-4TMM cell lines respond to catecholamine stimulation enhan-cing motility and invasion, and producing molecules closelyrelated to neoplastic progression. In keeping, we show thatNE and E are able to elicit activation of p42/p44 and p38MAPKs, acknowledged to have mandatory roles for cellgrowth, survival and invasive ability, in both primary andmetastatic cell lines. This observation is in agreement withthe findings of Pak et al.,23 also indicating the Ras-MAPKs
pathway as a target of b-adrenoceptors. These data could beof striking interest in order to find new strategies formelanoma treatment. In fact, Meier et al.24 showed thatcombined targeting of the p42/p44 and Akt signallingpathways significantly inhibited growth and enhancedapoptosis in melanoma cell cultures.
Moreover, NE and E exhibited a diverse stimulation ca-pacity on the two cell lines. Concerning the invasion assay,the primary cell line responded to NE (and, at a lesser degree,to E), while the metastatic cell line showed a clear reactiononly to E. The inhibition induced by ilomastat stronglysuggests a proteolytic degradation of Matrigel and theprobable intervention of MMPs. In fact, we demonstrate thatMMP-2, rather than MMP-9, is produced at a significantlyhigher level compared with baseline, by A375 cells under NEstimulus and by Hs29-4T cells under E stimulation. Fur-thermore, our data show that both NE and E are able to elicit
Figure 4 Expression of b2-AR in melanoma cell lines and analysis of the signalling pathway activated by NE stimulation. (a) Analysis of b2-AR
expression by immunoblot in primary (A375) and metastatic (Hs29-4T) melanoma cell lines. (b) Amount of ADRB2 mRNA by real-time PCR. The amount
of target was given by the 2�DDct calculation and was reported as 2�DDct. Immunoblot and real-time PCR are the mean of three different experiments.
(c, d) Analysis of activation of PKA. Melanoma cell lines were serum-deprived overnight and then stimulated with NE (1 mM) for the indicated period
and an immunoblot analysis for the detection of the phosphorylation level of p-PKA was shown. Actin immunoblot was used for normalization. The bar
graph below represents the phosphorylation level of PKA in four different experiments. *Po0.005.
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sustained activation of MMP-2 and MMP-9 both in primaryand metastatic cell lines. Thus, increased motility of mela-noma cells seems to be due to a proteolytic invasive capacity,typical of a mesenchymal phenotype,25,26 and catecholamines
seem able to influence MMPs activity both at a transcrip-tional and at a post-translational level. The migration ofneoplastic cells appears to be increased through activation ofb-ARs, because it is completely abolished by propranolol.
Figure 5 Analysis of activation of p42/p44 and p38 MAPK. Melanoma cell lines were serum-deprived overnight and then stimulated with E (a) or NE
(1 mM) (b) for the indicated period and an immunoblot analysis for the detection of the phosphorylation level of MAPKs were shown. Total p42/p44
and p38 MAPK immunoblot were used for normalization. The bar graphs below represent the phosphorylation level of MAPKs in four different
experiments. *Po0.005.
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With regard to the production of cytokine transcripts,A375 cells significantly increase levels of IL-6 and VEGFunder NE and E challenge, whereas Hs29-4T cells increaseIL-6 expression under NE stimulus, and produce significantamounts of VEGF, especially under E activation. Concerningthe expression of IL-8, the primary cell line responded to
both NE and E at a significantly higher degree compared withthe metastatic one, and both cell lines reacted more intenselyto NE. This result is in agreement with the angiogenic role ofIL-8, as de novo angiogenesis is particularly useful for primarytumours to escape the hostile microenvironment and dis-seminate metastasis.27 In addition, the catecholamine-induced
Figure 6 Effects of E and NE stimulation on melanoma cell lines invasion. Melanoma cell lines were serum-deprived overnight and then seeded in the
upper Boyden chamber for assay their invasion. (a, b) NE (a) or E (b) (1mM), in the presence or absence of ilomastat (50 mmol/l) or propranolol (1mM),
were added in the upper Boyden chamber. Bar graphs represent the mean of migrated cells counted in six different fields for each experiment.
*Po0.005 vs untreated. (c, d). Expression of MMP-2 (c) and MMP-9 (d) mRNA by real-time PCR. Melanoma cell lines were serum-deprived overnight and
then stimulated with E or NE (1mM) for 24 h. The amount of target, normalized to the GAPDH mRNA amounts, was given by the 2�DDCT calculation
and was reported as arbitrary units (a.u.). The graphs report data as the mean of three independent assays. (e, f) MMP-2 and MMP-9 activity assay.
Melanoma cell lines were serum-deprived overnight and then stimulated with NE or E (1 mM) for 24 h. The media obtained were then tested for MMPs
activity with a fluorimetric kit, following the manufacturer’s instructions (see Materials and Methods). Data are presented as RFU vs concentration of
test compounds. The graphs report data as the mean of four independent experiments. *Po0.005.
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IL-8 enhancement is in agreement with the described IL-8stimulation produced by NE in prostate cancer.10
We do not know exactly why such a discrepancy existsbetween primary and metastatic cell line response, but it ispossible that at least in part this difference is due to a higherexpression of b2-ARs, assessed as protein and transcript, onthe PM cell line. Another possibility is that IL-6 and IL-8,whose expression was associated with early malignancy ofmelanoma in vivo28 are actually secreted more efficiently by acell line derived from a PM.
In vitro experiments clearly show that catecholamines canaugment the malignant behaviour of melanoma cells affect-ing both invasion capacity and cytokine production.
Our in vitro experiments suggest that some pro-metastaticloops could work in melanoma in vivo too. We demonstratedthat IL-6 and NE in melanoma cells can activate dermal fi-broblasts towards a myofibroblastic phenotype, identified bya-SMA expression.21 It is well known that stromal fibroblastswithin tumours undergo a process, commonly calledmesenchymal–mesenchymal transition to myofibroblasts,leading them to achieve a more contractile phenotype andallowing a cross talk with tumour cells dramatically in-creasing their aggressiveness.29,30 In turn, activated fibroblastscan secrete other pro-metastatic cytokines, such as VEGF,31
capable of inducing further tumour angiogenesis. Inaddition, in our experiments, VEGF can increase,particularly when associated with NE, melanoma cellmigration and hence invasion capacity.
Previous data support the hypothesis that various types ofstress, such as surgical procedure and the immediate postoperative period, or neuroendocrine stress due to psycho-social factors, can stimulate tumour progression both inanimals and humans.10 This seems to be true also formelanoma at least in in vivo models.32,33
Our work provides evidence that stress hormones like NEand E can significantly stimulate the malignancy of mela-noma cells at various levels and that b-ARs, likely involved inthis response, are largely expressed in melanoma cell linesand cutaneous melanomas. It is the first time, to ourknowledge, that b-ARs are demonstrated in a large series ofhuman cutaneous melanocytic lesions, and even in melano-cytic naevi, suggesting a potential influence of catechola-mines also in benign counterparts. Moreover, the detection ofb2-AR also in the stromal cells of melanoma microenviron-ment implies further possible effects of catecholamines onmelanoma progression. Consequently, it is possible that theinteraction catecholamines-b-ARs could have a dramatic roleduring the clinical course of melanoma patients. The efforts
Figure 7 Expression of VEGF, IL-6 and IL-8 in E- and NE-treated melanoma cell lines. Melanoma cell lines were serum-deprived overnight and then
stimulated with E or NE (1 mM). Total RNA was used for the amplification of mRNA of IL-6 (a), VEGF (b) and IL-8 (c), using as housekeeping gene GAPDH
mRNA. The amount of target, normalized to the GAPDH mRNA amounts, was given by the 2�DDCT calculation and was reported as arbitrary units (a.u.).
The graphs report data as mean of three independent assays. *Po0.005.
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to understand molecular events underlying such an interac-tion can be very useful for indicating new targets in therapy.
ACKNOWLEDGEMENTS
This study was financially supported by fundings from Fondazione Cassa di
Risparmio di Pistoia e Pescia (Pistoia, Italy) and from Fondazione Banche di
Pistoia e Vignole per la Cultura e lo Sport (Quarrata, Pistoia, Italy). These
sponsors were not involved in any part of the study or in manuscript
preparation.
DISCLOSURE/CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Figure 8 Synergy among NE, cytokines and tumour microenvironment.
(a) A375 primary melanoma cells were serum-deprived overnight and
then seeded in the upper Boyden chamber for assaying their invasion.
NE (1 mM), IL-6 (50 ng/ml), VEGF (20 ng/ml), in combination with NE or
alone, were added in the upper Boyden chamber. Bar graph represents
the mean of migrated cells counted in six different fields for each
experiment. *Po0.005 vs untreated. (b) Analysis of human dermal
fibroblasts (HDFs) activation state, through evaluation of a-SMA
expression, after treatment with conditioned medium (CM) from NE/IL-6/
VEGF-treated A375 cells. A375 cells were grown to sub-confluence and
treated for 24 h with 1 mM NE, IL-6 (50 ng/ml) or VEGF (20 ng/ml). Fresh
serum-free medium was added for an additional 24 h before collection of
CM, in order to obtain CM free from NE, IL-6 or VEGF (but conditioned
by their earlier administration). HDFs were then incubated with the
obtained CM for 24 h and then used for western blot analysis of a-SMA
expression. Actin immunoblot was used for normalization. The blot is
representative of three different experiments.
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1
Quando ti metterai in viaggio per Itaca
devi augurarti che la strada sia lunga,
fertile in avventure e in esperienze.
I Lestrigoni e i Ciclopi
o la furia di Nettuno non temere,
non sarà questo il genere di incontri
se il pensiero resta alto e un sentimento
fermo guida il tuo spirito e il tuo corpo.
In Ciclopi e Lestrigoni, no certo,
nè nell’irato Nettuno incapperai
se non li porti dentro
se l’anima non te li mette contro.
Devi augurarti che la strada sia lunga.
Che i mattini d’estate siano tanti
quando nei porti - finalmente e con che gioia -
toccherai terra tu per la prima volta:
negli empori fenici indugia e acquista
madreperle coralli ebano e ambre
tutta merce fina, anche profumi
penetranti d’ogni sorta; più profumi inebrianti che puoi,
va in molte città egizie
impara una quantità di cose dai dotti.
Sempre devi avere in mente Itaca -
raggiungerla sia il pensiero costante.
Soprattutto, non affrettare il viaggio;
fa che duri a lungo, per anni, e che da vecchio
metta piede sull’isola, tu, ricco
dei tesori accumulati per strada
senza aspettarti ricchezze da Itaca.
2
Itaca ti ha dato il bel viaggio,
senza di lei mai ti saresti messo
sulla strada: che cos’altro ti aspetti?
E se la trovi povera, non per questo Itaca ti avrà deluso.
Fatto ormai savio, con tutta la tua esperienza addosso
già tu avrai capito ciò che Itaca vuole significare.
(Itaca – Konstantinos Kavafis)
